RELATED APPLICATIONS
TECHNICAL FIELD
[0002] The presently-disclosed subject matter relates to fluorescent compounds. In particular,
the presently-disclosed subject matter relates to azetidine-substituted polycyclic
chemical fluorophores as well as method for using the same.
INTRODUCTION
[0003] Fluorescence microscopy enables the imaging of specific molecules inside living cells.
This technique relies on the precise labeling of biomolecules with bright, photostable
fluorescent dyes. Genetically encoded fluorophores, such as green fluorescent protein
(GFP), are the mainstay of fluorescence imaging, allowing labeling with genetic specificity.
However, these proteinous dyes lack the requisite photostability for many applications
such as single-molecule imaging experiments. Over the past two decades, a number of
alternative labeling strategies have been developed that combine the genetic specificity
of fluorescent proteins with the favorable photophysics of small molecule fluorophores.
Attractive alternatives include FlAsH, enzyme-based self-labeling tags (
e.g., SnapTag and HaloTag), electrophilic ligand-receptor pairs (
e.g., TMPTag and coumarin-PYP), and lipoic acid ligase variants. Self-labeling tags allow
the labeling of a specific protein fusion with diverse synthetic fluorophores. Self-labeling
tags have enabled numerous imaging experiments inside living cells.
[0004] Although the general collection of chemical dyes is extensive, relatively few exhibit
the cell permeability needed for intracellular labeling. Thus, the available palette
of intracellular self-labeling tag ligands has been limited to classic, net neutral
fluorophores based on coumarin and rhodamine scaffolds, which exhibit membrane permeability
and rapid labeling kinetics, but suboptimal brightness and photostability. Previous
campaigns to improve dye performance (e.g., Cy, Alexa Fluor) involved substantial
modifications such as structural rigidification and addition of sulfonate groups.
These efforts resulted in highly polar, cell-impermeant dyes, useful
in vitro or on the cell exterior, but incompatible with live-cell intracellular applications.
[0005] Despite the compatibility of self-labeling tags and rhodamine dyes, little work has
been done to optimize this fluorophore class for live-cell labeling experiments. Previous
efforts have focused on increasing water solubility along with fluorescence brightness
and photostability, often through significant structural modifications. Such dyes
function for
in vitro and extracellular applications, but are too polar to passively enter cells.
[0006] Accordingly, there remains a need for compounds that are easy to synthesize, display
improved brightness, and exhibit appropriate cell permeability. There remains a need
for compounds that can function as self-labeling tags
in vivo.
SUMMARY
[0007] The presently-disclosed subject matter, as embodied and broadly described herein,
in one aspect, relates to compounds useful as fluorescent tags, and methods of using
the compounds to image one or more target substances, possibly in live cells. In some
embodiments the present compounds are azetidine-substituted derivatives of known fluorescent
tags. In some embodiments the present azetidine-substituted compounds can exhibit
greater quantum yields relative to their original parent compounds.
[0008] Compounds of the present invention are of the formula:

wherein:
each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR2, SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Q is selected from CR(2), NR, O, S, SiR(2), and Se;
W is selected from C and N;
X is selected from a lone pair of electrons, H, alkyl, aryl, halogen, CN, OH, O(alkyl),
O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, X being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Y is selected from CR(2), C(O)NR2, NR, O, and S; and
Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR2, PO3H2, SO3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H,
or wherein Z and Y, taken together with the atoms to which they are bonded, can form
a 5-7 membered ring which is unsubstituted or substituted with (a) one or more additional
heteroatoms selected from N, O and S or/and one or more substituents selected from
halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl or (b) an azetidine group;
wherein amine is independently selected from NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2; and
wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in claim
1.
[0009] Further disclosed are compounds of the formula:

wherein:
each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR2, SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Q is selected from CR(2), NR, O, S, SiR(2), and Se;
W is selected from C and N;
M is selected from CR(2), C(O), SO2 and PO2;
L is selected from O, S, NR, and CN2, wherein optionally L and W, taken together with the atoms to which they are bonded,
can form a substituted or unsubstituted 5-7 membered ring;
U and V are independently selected from H, alkyl, halogen, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, alkyl being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and/or SO3H, or wherein U and V, taken together with the atoms to which they are bonded, can
form a substituted or unsubstituted 5-7 membered ring;
Y is selected from CR(2), C(O)NR2, NR, O, and S; and
Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H,
or wherein Z and Y, taken together with the atoms to which they are bonded, can form
a 5-7 membered ring which is unsubstituted or substituted with (a) one or more additional
heteroatoms selected from N, O and S or/and one or more substituents selected from
halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl or (b) an azetidine group;
wherein amine is independently selected from NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2; and
wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in claim
1.
[0010] Embodiments of the presently-disclosed subject matter may be used in a kit. The kit
can include any of the compounds described herein and a binding element that binds
the compounds reversibly or irreversibly. The binding element may include a protein.
The kit may include a compound having the formula:

wherein:
each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR2, SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Q is selected from CR(2), NR, O, S, SiR(2), and Se;
W is selected from C and N;
X is selected from a lone pair of electrons, H, alkyl, aryl, halogen, CN, OH, O(alkyl),
O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, X being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Y is selected from CR(2), C(O)NR2, NR, O, and S; and
Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR2, PO3H2, SO3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H,
or wherein Z and Y, taken together with the atoms to which they are bonded, can form
a 5-7 membered ring which is unsubstituted or substituted with (a) one or more additional
heteroatoms selected from N, O and S or/and one or more substituents selected from
halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl or (b) an azetidine group;
wherein amine is independently selected from NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2; and
wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in claim
1; and
further comprising a binding element that binds the compound, optionally reversibly
or irreversibly.
[0011] The kits may include a compound of the formula:

wherein:
each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR2, SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Q is selected from CR(2), NR, O, S, SiR(2), and Se;
W is selected from C and N;
M is selected from CR(2), C(O), SO2 and PO2;
L is selected from O, S, NR, and CN2, wherein optionally L and W, taken together with the atoms to which they are bonded,
can form a substituted or unsubstituted 5-7 membered ring;
U and V are independently selected from H, alkyl, halogen, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, alkyl being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and/or SO3H, or wherein U and V, taken together with the atoms to which they are bonded, can
form a substituted or unsubstituted 5-7 membered ring;
Y is selected from CR(2), C(O)NR2, NR, O, and S; and
Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl or (b) an azetidine group;
wherein amine is independently selected from NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2; and
wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in claim
1; and
further comprising a binding element that binds the compound, optionally reversibly
or irreversibly.
[0012] Embodiments of the presently-disclosed subject matter also include a method for detecting
a target substance, comprising contacting a sample, which is suspected or known as
having the target substance, with any of the compounds described herein, and then
detecting an emission light from the compound, the emission light indicating the presence
of the target substance.
[0013] The method includes contacting a sample with a compound that selectively binds a
target substance, the compound being of the formula:

wherein:
each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR2, SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Q is selected from CR(2), NR, O, S, SiR(2), and Se;
W is selected from C and N;
X is selected from a lone pair of electrons, H, alkyl, aryl, halogen, CN, OH, O(alkyl),
O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, X being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Y is selected from CR(2), C(O)NR2, NR, O, and S; and
Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR2, PO3H2, SO3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl or (b) an azetidine group;
wherein amine is independently selected from NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2; and
wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in claim
1; and
detecting an emission light from the compound, the emission light indicating the presence
of the target substance.
[0014] Some methods include contacting a sample with a compound of the formula:

wherein:
each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR2, SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO3H2, and/or SO3H;
Q is selected from CR(2), NR, O, S, SiR(2), and Se;
W is selected from C and N;
M is selected from CR(2), C(O), SO2 and PO2;
L is selected from O, S, NR, and CN2, wherein optionally L and W, taken together with the atoms to which they are bonded,
can form a substituted or unsubstituted 5-7 membered ring;
U and V are independently selected from H, alkyl, halogen, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, alkyl being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and/or SO3H, or wherein U and V, taken together with the atoms to which they are bonded, can
form a substituted or unsubstituted 5-7 membered ring;
Y is selected from CR(2), C(O)NR2, NR, O, and S; and
Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, and SO3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO2, CHO, COOH, C(O)NR2, COO(alkyl), COO(aryl), PO3H2, SO3H, and alkyl or (b) an azetidine group;
wherein amine is independently selected from NH2, NH(alkyl), NH(aryl), N(alkyl)2, and N(aryl)2; and
wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in claim
1; and
detecting an emission light from the compound, the emission light indicating the presence
of the target substance.
[0015] Additional advantages of the invention will be set forth in part in the description
which follows, and in part will be obvious from the description, or can be learned
by practice of the invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly pointed out in the
appended claims. It is to be understood that both the foregoing general description
and the following detailed description are exemplary and explanatory only and are
not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The novel features of the subject matter of the present disclosure are set forth
with particularity in the appended claims. A better understanding of the features
and advantages of the presently disclosed subject matter will be obtained by reference
to the following detailed description that sets forth illustrative embodiments, in
which the principles of the present disclosure are used, and the accompanying drawings
of which:
Figure 1 includes a Jabloński diagram showing the process of twisted internal charge transfer
(TICT).
Figure 2 includes a plot showing the normalized absorption (abs) and fluorescence emission
(fl) spectra for tetramethylrhodamine and JF549.
Figure 3 includes a plot showing the normalized absorbance versus dielectric constant (εr) for tetramethylrhodamine and JF549.
Figure 4 includes a confocal maximum projection image of a nucleus from a live, washed HeLa
cell expressing HaloTag-H2B and incubated with JF549-HaloTag ligand; scale bar = 5 µm.
Figure 5 includes a whisker plot showing a comparison of brightness (n > 4,000) and track
length (n > 500) of HaloTag-H2B molecules labeled with JF549-HaloTag ligand or tetramethylrhodamine-HaloTag ligand, where the cross indicates
the mean and the whiskers span the 10-90 percentile.
Figure 6 includes a dSTORM fluorescence microscopy image of a fixed U2OS cell expressing HaloTag-H2B and
labeled with JF549-HaloTag ligand. The mean localization error was 17.2 nm, the median localization
error was 14.1 nm; scale bar = 5 µm.
Figure 7 includes dSTORM and wide-field (inset) fluorescence microscopy images of a fixed U2OS cell expressing
HaloTag-H2B and labeled with TMR-HaloTag ligand. The mean localization error was 19.2
nm, the median localization error was 17.0 nm; scale bar = 5 µm.
Figure 8 includes a plot showing normalized distributions of the localization errors for imaging
experiments using the JF549-HaloTag ligand (Figure 6) and the TMR-HaloTag ligand (Figure 7).
Figure 9 includes a dSTORM fluorescence microscopy image of the nucleus of a live HeLa cell expressing
HaloTag-H2B and labeled with JF549-HaloTag ligand; scale bar = 5 µm.
Figure 10 includes a dSTORM fluorescence microscopy image of a fixed U2OS cell nucleus expressing HaloTag-H2B
and labeled with JF646-HaloTag ligand. The mean localization error was 11.1 nm, the median localization
error was 8.4 nm; scale bar = 5 µm.
Figure 11 includes a dSTORM fluorescence microscopy image of a fixed U2OS cell expressing HaloTag-H2B and
labeled with SiTMR-HaloTag ligand. The mean localization error was 11.9 nm, the median
localization error was 9.0 nm; scale bar = 5 µm.
Figure 12 includes a plot showing normalized distributions of the localization errors for imaging
experiments using the JF646-HaloTag ligand (Figure 10) and the SiTMR-HaloTag ligand (Figure 11).
Figure 13 includes a wide-field fluorescence microscopy image of a live HeLa cell expressing
HaloTag-tubulin and labeled with JF646-HaloTag ligand.
Figure 14 includes a dSTORM microscopy image of a live HeLa cell expressing HaloTag-tubulin and labeled
with JF646-HaloTag ligand. The mean localization error was 9.23 nm; the median localization
error was 7.14 nm.
Figure 15 includes a plot showing line scan intensity in the wide-field image (Figure 13) and dSTORM image (Figure 14) as a function of line length.
Figure 16 includes a plot of the absorbance spectrum of SiTMR-HaloTag ligand (5 µM) in the
absence (-HT) and presence (+HT) of excess HaloTag protein.
Figure 17 includes a plot of the absorbance spectrum of JF646-HaloTag ligand (5 µM) in the absence (-HT) and presence (+HT) of excess HaloTag protein.
Figure 18 includes a wide-field fluorescence microscopy image of a live HeLa cell transfected
with H2B-HaloTag, incubated with SiTMR-HaloTag ligand (100 nM), and imaged without
intermediate washing steps; dashed line indicates cellular boundary; scale bars: 10
µm.
Figure 19 includes a wide-field fluorescence microscopy image of a live HeLa cell transfected
with H2B-HaloTag, incubated with JF646-HaloTag ligand (100 nM), and imaged without intermediate washing steps; dashed line
indicates cellular boundary; scale bars: 10 µm.
Figure 20 includes a plot of line scan intensity in Figure 18 as a function of line length.
Figure 21 includes a plot of line scan intensity in Figure 19 as a function of line length.
Figure 22 includes examples of wide-field fluorescence microscopy images of live unwashed HeLa
cells expressing HaloTag-H2B and incubated with 100 nM of either JF646-HaloTag ligand (top row) or SiTMR-HaloTag ligand (bottom row).
Figure 23 includes a rendering of single-molecule trajectories of SnapTag-TetR-JF549 conjugate from JF549-SnapTag ligand overlaid on a dSTORM H2B image of HaloTag-H2B labeled with JF646-HaloTag ligand in the nucleus of a live U2OS cell; scale bar = 5 µm.
Figure 24 includes an image showing the overlay of the dSTORM image of H2B and regions of fast TetR diffusivity (2-10 µm2 s-1; yellow) and slow TetR diffusivity (< 2 µm2 s-1; blue) in a live U2OS cell.
Figure 25 includes normalized distributions of the apparent diffusion coefficients (Dapp) of SnapTag-TetR that colocalize with HaloTag-H2B (black) or do not colocalize with
HaloTag-H2B (gray).
Figure 26 includes a wide-field fluorescence microscopy image showing fluorescence of DRAQ5
nuclear staining in live HeLa cells expressing SnapTag-H2B and labeled with DRAQ5
and Snap-Cell 430; scale bar = 50 µm.
Figure 27 includes a wide-field fluorescence microscopy image showing fluorescence of DRAQ5
nuclear staining in live HeLa cells expressing SnapTag-H2B and labeled with DRAQ5
and azetidinyl-coumarin-SnapTag ligand; scale bar = 50 µm.
Figure 28 includes a wide-field fluorescence microscopy image showing fluorescence of Snap-Cell
430-labeled SnapTag-H2B in live HeLa cells expressing SnapTag-H2B and labeled with
DRAQ5 and Snap-Cell 430; scale bar = 50 µm .
Figure 29 includes a wide-field fluorescence microscopy image showing fluorescence of azetidinyl-coumarin-labeled
SnapTag-H2B in live HeLa cells expressing SnapTag-H2B and labeled with DRAQ5 and azetidinyl-coumarin-SnapTag
ligand; scale bar = 50 µm .
Figure 30 includes a plot showing the quantification of the median nuclear fluorescence above
background coumarin label in cells when labeled with Snap-Cell 430 or azetidinyl-coumarin-SnapTag
ligand (n = 50, error bars, s.e.m.).
Figure 31 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 32 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 33 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 34 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 35 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 36 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 37 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
Figure 38 includes an exemplary synthetic scheme for synthesizing a compound in accordance
with an embodiment of the presently-disclosed subject matter.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0017] The details of one or more embodiments of the presently-disclosed subject matter
are set forth in this document. Modifications to embodiments described in this document,
and other embodiments, will be evident to those of ordinary skill in the art after
a study of the information provided in this document. The information provided in
this document, and particularly the specific details of the described exemplary embodiments,
is provided primarily for clearness of understanding and no unnecessary limitations
are to be understood therefrom. In case of conflict, the specification of this document,
including definitions, will control.
[0018] The presently-disclosed subject matter includes compounds that have utility as fluorophores
(e.g., fluorescent dyes). The present compounds can be utilized as fluorescent probes
to observe and characterize the location and/or concentration of particular substances.
In this regard, the terms "probe," "dyes," "tags," and the like are used interchangeably
herein to refer to compounds comprising a fluorophore moiety which is selective for
and/or is bonded to a binding element that is selective for a target substance. The
probes can emit an emission light, which can be used to determine the presence of
and/or measure the quantity of the target substance. In this respect, the presently-disclosed
subject matter also includes methods for using the present compounds and their intermediates,
as well as methods for preparing such compounds and the their intermediates.
Definitions
[0019] Unless defined otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which the
presently-disclosed subject matter belongs. Although any methods, devices, and materials
similar or equivalent to those described herein can be used in the practice or testing
of the presently-disclosed subject matter, representative methods, devices, and materials
are now described.
[0020] Following long-standing patent law convention, the terms "a", "an", and "the" refer
to "one or more" when used in this application, including the claims. Thus, for example,
reference to "a compound" includes a plurality of such compounds, and so forth.
[0021] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties
such as reaction conditions, and so forth used in the specification and claims are
to be understood as being modified in all instances by the term "about". Accordingly,
unless indicated to the contrary, the numerical parameters set forth in this specification
and claims are approximations that can vary depending upon the desired properties
sought to be obtained by the presently-disclosed subject matter.
[0022] As used herein, the term "about," when referring to a value or to an amount of mass,
weight, time, volume, concentration or percentage is meant to encompass variations
of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in
some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from
the specified amount, as such variations are appropriate to perform the disclosed
method.
[0023] As used herein, ranges can be expressed as from "about" one particular value, and/or
to "about" another particular value. It is also understood that there are a number
of values disclosed herein, and that each value is also herein disclosed as "about"
that particular value in addition to the value itself. For example, if the value "10"
is disclosed, then "about 10" is also disclosed. It is also understood that each unit
between two particular units are also disclosed. For example, if 10 and 15 are disclosed,
then 11, 12, 13, and 14 are also disclosed.
[0024] The term "absorption wavelength" as used herein refers to the wavelength of light
capable of being absorbed by a compound in order to excite the compound to emit a
light. The light emitted from a compound that has been excited with an absorption
light will have an "emission wavelength."
[0025] As used herein, the term "derivative" refers to a compound having a structure derived
from the structure of a parent compound (e.g., a compounds disclosed herein) and whose
structure is sufficiently similar to those disclosed herein and based upon that similarity,
would be expected by one skilled in the art to exhibit the same or similar activities
and utilities as the claimed compounds, or to induce, as a precursor, the same or
similar activities and utilities as the claimed compounds.
[0026] As used herein, the term "protein" means any polymer comprising any of the 20 protein
amino acids, regardless of its size. Although "polypeptide" is often used in reference
to relatively large proteins, and "peptide" is often used in reference to small proteins,
usage of these terms in the art overlaps and varies. The term "protein" as used herein
refers to peptides, polypeptides and proteins, unless otherwise noted.
[0027] The term "selectively bind" is used herein to refer to the property of an atom, moiety,
and/or molecule preferentially being drawn to or binding a particular compound. In
some instances the atom, moiety, and/or molecule selectively binds to a particular
site on a compound, such as an active site on a protein molecule.
[0028] The term "detect" is used herein to refer to the act of viewing, imagining, indicating
the presence of, measuring, and the like a target substance based on the light emitted
from the present compounds. More specifically, in some instances the present compounds
can be bound to a target substance, and, upon being exposed to an absorption light,
will emit an emission light. The presence of an emission light can indicate the presence
of a target substance, whereas the quantification of the light intensity can be used
to measure the concentration of a target substance.
[0029] The term "target substance" refers to a substance that is selectively bound directly
by the presently-disclosed compounds and/or indirectly by a molecule that is bound
to the present compound. A target substances can include, but is not limited to, a
protein, carbohydrates, polysaccharide, glycoprotein, hormone, receptor, antigen,
antibody, virus, substrate, metabolite, inhibitor, drug, nutrient, growth factor,
and the like. In some embodiments the target substance refers to an entire molecule,
and in other embodiments the target substances refers to a site on a molecule, such
as a binding site on a particular protein.
[0030] As used herein, the term "substituted" is contemplated to include all permissible
substituents of organic compounds. In a broad aspect, the permissible substituents
include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic,
and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents
include, for example, those described below. The permissible substituents can be one
or more and the same or different for appropriate organic compounds. For purposes
of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents
and/or any permissible substituents of organic compounds described herein which satisfy
the valences of the heteroatoms. This disclosure is not intended to be limited in
any manner by the permissible substituents of organic compounds.
[0031] Also, the terms "substitution" or "substituted with" include the implicit proviso
that such substitution is in accordance with permitted valence of the substituted
atom and the substituent, and that the substitution results in a stable compound,
e.g., a compound that does not spontaneously undergo transformation such as by rearrangement,
cyclization, elimination, etc. Unless stated otherwise, all chemical groups described
herein include both unsubstituted and substituted varieties.
[0032] In defining various terms, "A
1," "A
2," "A
3," and "A
4" are used herein as generic symbols to represent various specific substituents. These
symbols can be any substituent, not limited to those disclosed herein, and when they
are defined to be certain substituents in one instance, they can, in another instance,
be defined as some other substituents.
[0033] Where substituent groups are specified by their conventional chemical formula written
from left to right, they equally encompass the chemically identical substituents that
would result from writing the structure from right to left. For instance, --CH
2O-- also encompasses recite -OCH
2-.
[0034] It should be understood that the bond types and locations in the chemical structures
provided herein may adapt depending on the substituents in the compound, even if not
specifically recited. For instance,-X- where X can be either C or N can refer to,
respectively, -CH2- or -NH-, where the lone pair of electrons on N is not illustrated.
Thus, even if not specifically illustrated, the chemical compounds described herein
include any hydrogen atoms, lone pair of electrons, and the like necessary for completing
a chemical structure.
[0035] The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon
group of 1 to 24 carbon atoms, such as methyl, ethyl,
n-propyl, isopropyl,
n-butyl, isobutyl,
s-butyl,
t-butyl,
n-pentyl, isopentyl,
s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl,
eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl
group can be branched or unbranched. The alkyl group can also refer to both substituted
or unsubstituted alkyls. For example, the alkyl group can be substituted with one
or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl,
alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described
herein. A "lower alkyl" group is an alkyl group containing from one to six (e.g.,
from one to four) carbon atoms.
[0036] Throughout the specification "alkyl" is generally used to refer to both unsubstituted
alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also
specifically referred to herein by identifying the specific substituent(s) on the
alkyl group. For example, the term "halogenated alkyl" specifically refers to an alkyl
group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine,
or iodine. The term "alkoxyalkyl" specifically refers to an alkyl group that is substituted
with one or more alkoxy groups, as described below. The term "alkylamino" specifically
refers to an alkyl group that is substituted with one or more amino groups, as described
below, and the like. When "alkyl" is used in one instance and a specific term such
as "alkylalcohol" is used in another, it is not meant to imply that the term "alkyl"
does not also refer to specific terms such as "alkylalcohol" and the like.
[0037] This practice is also used for other groups described herein. That is, while a term
such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties,
the substituted moieties can, in addition, be specifically identified herein; for
example, a particular substituted cycloalkyl can be referred to as,
e.g., an "alkylcycloalkyl." Similarly, a substituted alkoxy can be specifically referred
to as,
e.g., a "halogenated alkoxy," a particular substituted alkenyl can be,
e.g., an "alkenylalcohol," and the like. Again, the practice of using a general term, such
as "cycloalkyl," and a specific term, such as "alkylcycloalkyl," is not meant to imply
that the general term does not also include the specific term. The term "alkyl" is
inclusive of "cycloalkyl."
[0038] The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed
of at least three carbon atoms. Examples of cycloalkyl groups include, but are not
limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like.
The term "heterocycloalkyl" is a type of cycloalkyl group as defined above, and is
included within the meaning of the term "cycloalkyl," where at least one of the carbon
atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen,
oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can
be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can
be substituted with one or more groups including, but not limited to, optionally substituted
alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo,
or thiol as described herein.
[0039] In this regard, the term "heterocycle," as used herein refers to single and multi-cyclic
aromatic or non-aromatic ring systems in which at least one of the ring members is
other than carbon. Heterocycle includes pyridinde, pyrimidine, furan, thiophene, pyrrole,
isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including,
1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,thiadiazole, including, 1,2,3-thiadiazole,
1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole,
tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine,
pyrimidine, pyrazine, triazine, including 1,2,4-triazine and 1,3,5-triazine, tetrazine,
including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine,
tetrahydropyran, tetrahydrofuran, dioxane, and the like.
[0040] The terms "alkoxy" and "alkoxyl" as used herein to refer to an alkyl or cycloalkyl
group bonded through an ether linkage; that is, an "alkoxy" group can be defined as
-OA
1 where A
1 is alkyl or cycloalkyl as defined above. "Alkoxy" also includes polymers of alkoxy
groups as just described; that is, an alkoxy can be a polyether such as -OA
1-OA
2 or -OA
1-(OA
2)
a-OA
3, where "a" is an integer of from 1 to 200 and A
1, A
2, and A
3 are alkyl and/or cycloalkyl groups.
[0041] The term "alkenyl" as used herein is a hydrocarbon group of from 2 to 24 carbon atoms
with a structural formula containing at least one carbon-carbon double bond. The term
is include of linear and ring-forming (i.e., cycloakenyl) groups. Asymmetric structures
such as (A
1A
2)C=C(A
3A
4) are intended to include both the E and Z isomers. This can be presumed in structural
formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated
by the bond symbol C=C. The alkenyl group can be substituted with one or more groups
including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,
cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic
acid, ester, ether, haide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol,
as described herein.
[0042] The term "aryl" as used herein is a group that contains any carbon-based aromatic
group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene,
and the like. The term "aryl" also includes "heteroaryl," which is defined as a group
that contains an aromatic group that has at least one heteroatom incorporated within
the ring of the aromatic group. Examples of heteroatoms include, but are not limited
to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term "non-heteroaryl,"
which is also included in the term "aryl," defines a group that contains an aromatic
group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted.
The aryl group can be substituted with one or more groups including, but not limited
to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,
cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,
hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The
term "biaryl" is a specific type of aryl group and is included in the definition of
"aryl." Biaryl refers to two aryl groups that are bound together
via a fused ring structure, as in naphthalene, or are attached
via one or more carbon-carbon bonds, as in biphenyl.
[0043] The term "ring" as used herein refers to a substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl,
or substituted or unsubstituted heteroaryl. A ring includes fused ring moieties, referred
to as a fused ring system wherein a ring may be fused to one or more rings selected
from a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl in
any combination. The number of atoms in a ring is typically defined by the number
of members in the ring. For example, a "5- to 8-membered ring" means there are 5 to
8 atoms in the encircling arrangement. A ring can optionally include a heteroatom.
The term "ring" further includes a ring system comprising more than one "ring", wherein
each "ring" is independently defined as above.
[0044] Some of the unsaturated structures described herein, such as ring structures including
cycloalkyl and aryl, are illustrated with dashed bonds to signify the potential existence
of a resonance structure. Structures having dashed bonds are intended to reflect every
possible configuration of the structure, but does not necessarily imply that all possible
structures are in existence. It should be understood that the types of bonds (e.g.,
single bond, double bond) in such structures will vary depending on the atoms in the
structure as well as whether the structures are substituted with one or more additional
atoms or moieties.
[0045] The term "aldehyde" as used herein is represented by a formula -C(O)H. Throughout
this specification "C(O)" is a short hand notation for a carbonyl group,
i.e., C=O.
[0046] The terms "amine" or "amino" as used herein are represented by a formula NA
1A
2A
3, where A
1, A
2, and A
3 can be, independently, hydrogen or optionally substituted alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
In specific embodiments amine refers to any of NH
2, NH(alkyl), NH(aryl), N(alkyl)
2, and N(aryl)
2.
[0047] The term "carboxylic acid" as used herein is represented by a formula -C(O)OH.
[0048] The term "ester" as used herein is represented by a formula -OC(O)A
1 or -C(O)OA
1, where A
1 can be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,
cycloalkynyl, aryl, or heteroaryl group as described herein. The term "polyester"
as used herein is represented by a formula - (A
1O(O)C-A
2-C(O)O)
a- or -(A
1O(O)C-A
2-OC(O))
a-, where A
1 and A
2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,
alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and "a" is an integer
from 1 to 500. "Polyester" is as the term used to describe a group that is produced
by the reaction between a compound having at least two carboxylic acid groups with
a compound having at least two hydroxyl groups.
[0049] The term "halide" or "halogen" refers to at least one of the halogens selected from
fluorine, chlorine, bromine, and iodine.
[0050] The term "thiol" as used herein is represented by a formula -SH.
Compounds
[0051] The presently-disclosed subject matter includes compounds that are azetidine-substituted.
In certain embodiments the azetidine-substituted compounds are modified forms of compounds
comprising an electron-donating
N,N-dialkylamino motif for fluorescence. In such embodiments the
N,N-dialkyl group of the original parent compound is replaced with azetidine. Some unmodified
fluorophores comprising the
N,N-dialkylamino motif are prone to a nonradiative decay mechanism and/or exhibit modest
quantum yield. However, embodiments of the present compounds that include a substitution
of the dimethylamino group for an azetidine moiety can reduce or eliminate this nonradiative
decay pathway and increase the quantum yield values of the fluorophores relative to
corresponding non-azetidine-substituted compounds.
[0052] By virtue of having increased quantum yields, some embodiments of the present compounds
also exhibit brightness and photostability that is comparable or superior to corresponding
non-azetidine-substituted compounds. In some embodiments compounds with improved quantum
yield require lower illumination powers for biological imaging experiments, and are
less likely to undergo destructive relaxation pathways, resulting in higher photostability.
[0053] The properties of certain embodiments of the present compounds are superior and unexpected.
A planar structure can be beneficial for fluorescent emissions to occur in xanthenium
dyes and other similar structures. It had previously been thought that substitution
with lower rings, such as those having about 3 or 4 carbons, would compromise the
planar structure of the compounds. Specifically, modification with a four-membered
azetidine ring system is highly strained (26 kcal mol
-1) and intuitively was not believed not to be compatible with the planar delocalized
structures found in many fluorescent molecules.
[0054] The present inventors found that the novel azetidine-substitution described herein
surprisingly and unexpectedly retain and can even enhance the fluorescent characteristics
of the corresponding non-azetidine-substituted compounds.
[0055] Embodiments of the present azetidine-substituted compounds also comprise a structure
that can be less susceptible to undergo a twisted internal charge transfer (TICT).
This surprising and unexpected characteristic provides certain embodied compounds
with a high quantum yield, and in some instances a quantum yield that is higher than
that of the base non-azetidine-substituted compound.
[0056] It should be understood that the presently-disclosed azetidine-substitutions can
be performed on a wide variety of fluorophores, including known fluorophores. In some
instances the azetidine-substitution permit the base fluorophores to retain and/or
enhance their beneficial properties. For example, embodiment embodiments of the present
compounds include azetidine-substituted rhodamine compounds that retain or enhance
the brightness, photostability, and/or insensitivity to light of non-azetidine-substituted
rhodamine compounds.
[0057] Of the extant collection of chemical fluorophores, the rhodamine dyes are a useful
class for live-cell imaging with genetically encoded self-labeling tags. This utility
stems from the brightness, photostability, insensitivity to pH, and modifiable structure
of rhodamine dyes. The spectral characteristics of rhodamines can be controlled to
allow access to dyes with absorption maxima ranging from blue to the infrared. In
addition, rhodamine dyes exist in equilibrium between an "open," zwitterionic, quinoid
form and a "closed," lipophilic, lactone form. This dynamic amphipathicity makes rhodamine
dyes excellent ligands for live-cell labeling technologies; the dye efficiently traverses
the cellular membrane without detergents or chemical masking groups and excess ligand
can be rapidly washed away.
[0058] According to the invention, a compound of the following formula is provided:

wherein each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR
2, SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H; Q is selected from CR
(2), C(O)NR
2, NR, O, S, SiR
(2), and Se; W is selected from C and N; X is selected from a lone pair of electrons,
H, alkyl, aryl, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, X being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H; Y is selected from CR
(2), C(O)NR
2, NR, O, and S; and Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH,
S(alkyl), S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, SO
3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl or (b) an azetidine group; wherein amine is independently selected from
NH
2, NH(alkyl), NH(aryl), N(alkyl)
2, and N(aryl)
2; and wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in
claim 1.
[0059] In some embodiments wherein Y and Z, taken together with the atoms to which they
are bonded, form the 5-7 membered ring being substituted with one or more additional
heteroatoms selected from N, O and S or/and one or more substituents selected from
halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl. In specific embodiments wherein Y and Z, taken together with the atoms
to which they are bonded, form the 5-7 membered ring that is substituted with an unsubstituted
or substituted azetidine group.
[0060] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

wherein R' is selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl),
amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, SO
3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl),
C(O)NR
2, amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H. In other embodiments R' is selected from an azetidine moiety (group) that is unsubstituted
or is substituted with one or more of halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), C(O)NR
2, COO(aryl), PO
3H
2, SO
3H, and alkyl.
[0061] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0062] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0063] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0064] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0065] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0066] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0067] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0068] In some embodiments of the presently-disclosed subject matter, a compound can be
of the following formula:

[0069] In some embodiments of the presently-disclosed subject matter, X is a substituted
aryl.
[0070] In other embodiments, X can also be selected from, but is not limited to, H, C,

In some embodiments, W is N and X is a lone pair of electrons. In some embodiments
X can partially or wholly comprise a linker that is capable of binding the present
compounds to a binding element, as described herein. In other embodiments X can partially
or wholly comprise a binding element. The structures of X illustrated herein are provided
for illustrative purposes only, as X in some embodiments is dependent on the linker
that may be used in conjunction with a compound, the binding element that may be used
in conjunction with a compound, and/or the target substance to be detected by a compound.
[0072] The presently-disclosed subject matter also includes derivatives of any of the compounds
described herein.
[0073] The compounds described herein can contain one or more double bonds and, thus, potentially
can give rise to cis/trans (E/Z) isomers, as well as other conformational isomers.
Unless stated to the contrary, the invention includes all such possible isomers, as
well as mixtures of such isomers. Unless stated to the contrary, a formula with chemical
bonds shown only as solid lines and not as wedges or dashed lines contemplates each
possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers,
such as a racemic or scalemic mixture. Compounds described herein can contain one
or more asymmetric centers and, thus, potentially give rise to diastereomers and optical
isomers. Unless stated to the contrary, the present invention includes all such possible
diastereomers as well as their racemic mixtures, their substantially pure resolved
enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts
thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are
also included. During the course of the synthetic procedures used to prepare such
compounds, or in using racemization or epimerization procedures known to those skilled
in the art, the products of such procedures can be a mixture of stereoisomers.
[0074] As discussed herein, it should be understood that the presently-disclosed azetidine-substitutions
are generalizable and can be applied to a wide variety of compounds. Exemplary compounds
include azetidine-substituted rhodamine derivatives, azetidine-substituted coumarin
derivatives, azetidine-substituted rhodol derivatives, azetidine-substituted acridine
derivatives, azetidine-substituted oxazine derivatives, azetidine-substituted naphthalimide
derivatives, azetidine-substituted carborhodamine derivatives, azetidine-substituted
silarhodamine derivatives, and the like. Those of ordinary skill will recognize other
compounds capable of the presently-disclosed azetidine-substitutions. As described
above, these and other derivatives of the presently-disclosed subject matter can retain
and/or enhance the beneficial characteristics of the corresponding non-azetidine-substituted
compounds. For example, embodiments of azetidine-substituted compounds with minimal
structural changes can preserve the cell permeability and efficiency of intracellular
labeling of the original non-azetidine-substituted compound.
[0075] Furthermore, as described herein, the azetidine moieties can be substituted or unsubstituted.
Table 1 below describes embodiments of azetidinyl-rhodamines, azetidinyl-carborhodamines,
and azetidinyl-sila-rhodamines bearing substituents on the azetidine rings. It should
be understood that the azetidine moieties described in
Table 1 can be incorporated into any of the compounds described herein.
Table 1. Spectroscopic data for embodiments of azetidinyl-rhodamines, azetidinyl-carborhodamines,
and azetidinyl-sila-rhodamines.
NR2 |
X |
λmax (nm) |
ε (M-1 cm-1) |
λem (nm) |
ø |

|
O |
549 |
101,000 |
571 |
0.88 |

|
O |
550 |
110,000 |
572 |
0.83 |

|
O |
541 |
109,000 |
564 |
0.88 |

|
O |
542 |
111,000 |
565 |
0.57 |

|
O |
536 |
113,000 |
560 |
0.87 |
O |
525 |
94,000 |
549 |
0.91 |

|
O |
533 |
108,000 |
557 |
0.89 |

|
O |
545 |
108,000 |
568 |
0.87 |

|
O |
549 |
111,000 |
572 |
0.87 |

|
CMe2 |
608 |
99,000 |
631 |
0.67 |
CMe2 |
585 |
156,000a |
609 |
0.78 |

|
SiMe2 |
646 |
152,000b |
664 |
0.54 |

|
SiMe2 |
635 |
167,000a |
652 |
0.56 |
All measurements were taken in 10 mM HEPES pH 7.3 unless otherwise noted.
a Extinction coefficient measured in trifluoroethanol containing 0.1% (v/v) trifluoroacetic
acid.
b Extinction coefficient measured in ethanol containing 0.1% (v/v) trifluoroacetic
acid. |
[0076] The present compounds can have a broad range of absorption and emission properties.
Since the present azetidine-substitution can be performed on a variety of compounds,
embodiments of the present azetidine-substituted compounds can include absorption
wavelengths in the ultraviolet to the near-infrared spectrum. Specific embodiments
of the present compounds can include absorption wavelengths selected of about 100
nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or any
value therebetween. In some embodiments the compounds include an absorption wavelength
of more than about 1000 nm. Once activated, the present compounds can emit a detectable
emission light. The wavelength of the emission light can vary depending on the base
compound its substitutions, and in some embodiments the emission wavelength is a wavelength
of about 100 nm to about 1000 nm, including about 100 nm, 200 nm, 300 nm, 400 nm,
500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or any value therebetween.
[0077] Those of ordinary skill in the art will also appreciate that the present compounds
comprise both open and closed (e.g. lactone) forms of the present fluorophores. In
some instances the present azetidinyl fluorophores that possess ester substituents
can be deprotected through acid- or base-mediated conditions to generate azetidinyl
dyes with carboxylic acid handles. The scheme shown in
Figure 33 illustrates an exemplary fluorophore transitioning between a closed "lactone" form
and an open form having a carboxylic acid handle. In other embodiments the compounds
can transition from a closed form to an open form upon being exposed to an absorption
light. Thus, in some embodiments, the present compounds can be photo or chemically
activated in order to transition between closed and open forms. All of the compounds
described herein include both the closed form and open form for each fluorophore.
[0078] In some embodiments, both open and closed forms of embodiments of the presently-disclosed
compounds can be represented by the following formula:

wherein each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR
2, SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H; Q is selected from CR
(2), C(O)NR
2, NR, O, S, SiR
(2), and Se; W is selected from C and N; M is selected from CR
(2), C(O)NR
2, C(O), SO
2 and PO
2; L is selected from O, S, NR, CN
2, and C(O)NR
2, wherein optionally L and W, taken together with the atoms to which they are bonded,
can form a substituted or unsubstituted 5-7 membered ring; U and V are independently
selected from H, alkyl, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), C(O)NR
2, S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, alkyl being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR
2, SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H, or wherein U and V, taken together with the atoms to which they are bonded, can
form a substituted or unsubstituted 5-7 membered ring; Y is selected from CR
(2), C(O)NR
2, NR, O, and S; and Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH,
S(alkyl), S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, SO
3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl or (b) an azetidine group; wherein amine is independently selected from
NH
2, NH(alkyl), NH(aryl), N(alkyl)
2, and N(aryl)
2; and wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in
claim 1.
[0079] In some embodiments of such compounds, the compounds have a structure represented
by the following formula:

wherein R' is selected from halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl),
amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, SO
3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl),
amine, NO
2, CHO, COO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, or an azetidine group that is unsubstituted or substituted with one or more of
halogen, H, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, C(O)NR
2, COOH, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl..
[0080] In this regard, exemplary open form compounds of the presently-disclosed subject
matter have the following formula:

[0081] In some embodiments, the closed form of the presently-disclosed compounds include
the following formula:

[0082] In other embodiments, the closed form of the presently-disclosed compounds include
the following formula:

[0083] In some embodiments of U and V include a substituted aryl, and in some embodiments
the compound can be represented by the following formula:

wherein X' is selected from H, OH, alkyl, aryl, halogen, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, and SO
3H,
[0085] While the above structures are provided for illustrative purposes, those of ordinary
skill in the art will appreciate all the open and closed forms of the compounds disclosed
herein upon review of this paper.
Kits
[0086] The presently-disclosed subject matter may be used in kits comprising any of the
compound(s) described herein, packaged together with an appropriate binding element.
In some instances binding elements are referred to as ligands herein. The binding
element can bind the compound reversibly and/or irreversibly. The binding element
can be bound to the compound directly, or the binding element can be bound to the
compound indirectly. A compound can be bound to a binding element indirectly via a
linker, wherein the linker can include unsubstituted or substituted alkyl or the like.
Kits can further be provided with a linker for attachment to the present compounds.
[0087] The binding element can generally selectively bind a molecule or substance of interest
(i.e., target substance). Exemplary binding elements include, but are not limited
to, amino acid(s), a protein, an antibody or fragment thereof, an antigen, a polysaccharide,
a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a drug, a hormone,
a lipid, a synthetic polymer, a solid support, a polymeric microparticle, a cell,
a virus, an enzymatic substrate, and the like. or a virus. Binding elements can be
used to detect a molecule or substance to be observed and/or characterized, can indicate
a particular event has occurred, and/or can indicate the presence of another molecule
or substance (i.e., target substance).
[0088] A kit can comprise two or more different compounds in accordance with the presently-disclosed
subject matter. Such kits may be further provided with one or more bind elements,
wherein the compounds can by bound to the same or different binding elements. Each
of the compounds and/or binding elements may selectively bind different molecules,
particles, substances, or the like. Additionally or alternatively, the kit may comprise
two or more different compounds in accordance with the presently-disclosed subject
matter that have different absorption wavelengths and/or emission wavelengths, and
therefore can be practiced during multiplex procedures.
Methods of Use
[0089] The presently-disclosed subject matter further includes a method of using the compounds
described herein. In some embodiments the method comprises utilizing the compound
as a reporter for enzyme activity, as a fluorescent tag, as a sensor for a target
substance (an analyte), as an agent for imaging experiments, and/or as an imaging
agent for super-resolution microscopy.
[0090] Some embodiments of the presently-disclosed subject matter include methods for detecting
a target sample that comprise contacting a sample with a compound of the following
formula:

wherein each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR
2, SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H; Q is selected from CR
(2), NR, O, S, SiR
(2), and Se; W is selected from C and N; X is selected from a lone pair of electrons,
H, alkyl, aryl, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine,
NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, X being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H; Y is selected from CR
(2), C(O)NR
2, NR, O, and S; and Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH,
S(alkyl), S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, SO
3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl or (b) an azetidine group; wherein amine is independently selected from
NH
2, NH(alkyl), NH(aryl), N(alkyl)
2, and N(aryl)
2; and wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in
claim 1.
[0091] Alternatively or additionally, methods are disclosed for detecting a target sample,
the method comprises a step of contacting a sample with a compound of the following
formula:

wherein each R is independently selected from halogen, H, CN, OH, O(alkyl), O(aryl),
SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl, alkyl being optionally substituted with one or more heteroatoms independently
selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR
2, SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H; Q is selected from CR
(2), NR, O, S, SiR
(2), and Se; W is selected from C and N; M is selected from CR
(2), C(O), SO
2 and PO
2; L is selected from O, S, NR, and CN
2, wherein optionally L and W, taken together with the atoms to which they are bonded,
can form a substituted or unsubstituted 5-7 membered ring; U and V are independently
selected from H, alkyl, halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), C(O)NR
2, S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, alkyl being optionally substituted with one or more heteroatoms independently selected
from N, O, and S, halogen, OH, O(alkyl), O(aryl), C(O)NR
2, SH, S(alkyl), S(aryl), amine, NO
2, CHO, COO, COOH, COO(alkyl), COO(aryl), PO
3H
2, and/or SO
3H, or wherein U and V, taken together with the atoms to which they are bonded, can
form a substituted or unsubstituted 5-7 membered ring; Y is selected from CR
(2), C(O)NR
2, NR, O, and S; and Z is selected from H, halogen, CN, OH, O(alkyl), O(aryl), SH,
S(alkyl), S(aryl), amine, NO
2, CHO, COOH, COO(alkyl), COO(aryl), C(O)NR
2, PO
3H
2, SO
3H, aryl, and alkyl, alkyl and aryl being optionally substituted with one or more heteroatoms
independently selected from N, O, and S, halogen, OH, O(alkyl), O(aryl), SH, S(alkyl),
S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, and SO
3H, or wherein Z and Y, taken together with the atoms to which they are bonded, can
form a 5-7 membered ring which is unsubstituted or substituted with (a) one or more
additional heteroatoms selected from N, O and S or/and one or more substituents selected
from halogen, CN, OH, O(alkyl), O(aryl), SH, S(alkyl), S(aryl), amine, NO
2, CHO, COOH, C(O)NR
2, COO(alkyl), COO(aryl), PO
3H
2, SO
3H, and alkyl or (b) an azetidine group; wherein amine is independently selected from
NH
2, NH(alkyl), NH(aryl), N(alkyl)
2, and N(aryl)
2; and wherein the terms "alkyl", "aryl" and "5-7 membered ring" are as defined in
claim 1.
[0092] The presently-disclosed method for detecting a target substance can further comprise
a detecting step that includes detecting an emission light from the compound, the
emission light indicating the presence of the target substance.
[0093] In some embodiments the method for using the compounds further comprises exciting
the compound by exposing the compound to an absorption light that includes an absorption
wavelength. As described herein, the absorption light can include a of ultraviolet
light to near infrared light. In specific embodiments the absorption wavelength can
be in a range from 100 nm to 1000 nm, in a range of 200 nm to 800 nm, and/or in a
range of 450 nm to 650 nm. In some embodiments the absorption wavelength is about
100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1,000 nm.
[0094] In some embodiments the detecting step is performed by use of fluorescence spectroscopy
or by the naked eye. Thus, in some embodiments the detecting step is performed with
a microscope. In some embodiments the presence of a target substance can indicate
the occurrence or absence of a particular biological function, as will be appreciated
by those of ordinary skill in the art. In some embodiments the method is performed
in a live cell and/or subject.
[0095] Some embodiments of detection methods comprise contacting the sample with two or
more embodiments of compounds that are selective for different target substances.
Methods for detecting two or more target substances with two or more of the presently-disclosed
compounds are referred to herein as "multiplex" detection methods.
[0096] In some of the present multiplex methods, two or more distinct target substances
and/or two or more regions of one target substance are detected using two or more
probes, wherein each of the probes is labeled with a different embodiment of the present
compounds. The presently-disclosed compounds can be used in multiplex detection methods
for a variety of target substances.
[0097] In this regard, multiplex methods can comprise contacting the sample with a first
compound and a second compound in accordance with the presently-disclosed subject
matter. The first compound can be selective for a first target substance and can be
capable of emitting a first emission light, and the second compound can be selective
for a second target substance and can be of emitting a second emission light. The
detecting step includes detecting the first emission light that indicates the presence
of the first target substance and the second emission light that indicates the presence
of the second target substance., and then detecting a second emission light from the
compound. The second emission light can indicate the presence of a second target substance.
In some embodiments the emission wavelength and the second emission wavelength are
different form one another. This novel method thereby provides an efficient means
for detecting a plurality of different target substances in one substance simultaneously.
Methods of Synthesis
[0098] Also described is a method of producing a compound as described herein. Methods for
synthesizing embodiments of the presently-disclosed compounds generally include one
or more well-known synthesis steps. While certain methods for synthesizing the present
compounds are described herein, methods of synthesis should be not limited to the
methods described herein, as methods for synthesis can include any methods that would
be readily apparent to those of ordinary skill in the art.
[0099] The method may comprise forming the C(aryl)-N (e.g., C(aryl)-azetidine) bonds of
the compounds at a late stage in the synthesis method. Buchwald-Hartwig cross-coupling
of nitrogen nucleophiles with fluorescein ditriflates may be utilized. The method
may proceed under Buchwald-Hartwig conditions using Pd(OAc)
2, BINAP, and Cs
2CO
3 in toluene at about 100°C. Or the method may proceed utilizing Pd
2dba
3 with the active biaryl ligand XPhos, with Cs
2CO
3 as a base and dioxane as a solvent at about 80°C to about 100°C. Thus, Pd-catalyzed
cross-coupling with disparate
N-alkyl coupling partners may be used as a method for synthesizing the present compounds.
[0101] Furthermore, a non-limiting list of methods for synthesizing embodiments of the presently-disclosed
compounds are illustrated in the exemplary schemes shown in
Figures 31 to 38.
[0102] Figure 31 includes a general synthesis scheme for the preparation of exemplary dibromofluorans
and fluorescein ditriflates. Dibromofluorans and fluorescein diacetates possessing
carboxylic acid substituents can be protected as esters via acid-catalyzed Fischer
esterification or through reaction with the di-
tert-butyl acetal of DMF. The fluorescein diacetates can then be hydrolyzed with base;
the resulting substituted fluoresceins can be converted to fluorescein ditriflates
with trifluoromethanesulfonic anhydride.
[0103] Figure 32 includes a general synthesis scheme for the preparation of exemplary carbo- and silafluorescein
ditriflates. Reaction of TBS-protected anthrones and Si-anthrones with aryl Grignard
reagents can allow access to TBS-protected carbofluoresceins and silafluoresceins.
TBAF-mediated deprotection followed by reaction with trifluoromethanesulfonic anhydride
can afford carbofluorescein ditriflates and silafluorescein ditriflates.
[0104] Figure 33 includes a general synthesis scheme for the preparation of exemplary azetidinyl-rhodamines,
-carborhodamines, and -sila-rhodamines. Buchwald-Hartwig palladium-catalyzed C-N cross-coupling
of azetidines with dibromofluorans, fluorescein ditriflates, carbofluorescein ditriflates,
or silafluorescein ditriflates can directly afford azetidinyl fluorophores. The azetidinyl
fluorophores that possess ester substituents can be deprotected through acid- or base-mediated
conditions to generate azetidinyl dyes with carboxylic acid handles. The acids can
be further derivatized into N-hydroxysuccinimidyl (NHS) esters by reaction with DSC
or TSTU and subsequently reacted with amines to generate analogs with pendant amide
groups.
[0105] Figure 34 includes a general synthesis scheme for the preparation of exemplary azetidinyl-rhodols.
Buchwald-Hartwig palladium-catalyzed C-N cross-coupling of azetidines (1 equivalent)
with fluorescein ditriflates, carbofluorescein ditriflates, or silafluorescein ditriflates
can provide, upon hydrolysis of the remaining triflate moiety, azetidinyl-rhodols.
[0106] Figure 35 includes a general synthesis scheme for the preparation of exemplary fluorogenic
2-diazo-1-indanone dyes from azetidinyl-rhodamines, -carborhodamines, and -sila-rhodamines.
Reaction of azetidinyl-rhodamines with oxalyl chloride followed by (trimethylsilyl)diazomethane
can afford 2-diazo-1-indanones. Base-mediated hydrolysis of ester substituents can
yield 2-diazo-1-indanones with acid moieties, which can be further derivatized into
N-hydroxysuccinimidyl (NHS) esters by reaction with TSTU and reacted with amines to
generate analogs with pendant amide groups.
[0107] Figure 36 includes a general synthesis scheme for the preparation of exemplary azetidinyl-coumarins.
Coumarin triflates can be synthesized by reaction of 7-hydroxycoumarins with trifluoromethanesulfonic
anhydride or
N-phenyl-bis(trifluoromethanesulfonimide). Buchwald-Hartwig palladium-catalyzed C-N
cross-coupling of azetidines with coumarin triflates can yield azetidinyl-coumarins.
Alternatively, azetidinyl-phenols can be prepared by C-N cross-coupling of bromophenols
with azetidines. Vilsmeier-Haack formylation of azetidinyl-phenols can then provide
aldehydes that can be condensed with malonates to afford azetidinyl-coumarins. For
examples possessing ester substituents, base-mediated hydrolysis can yield azetidinyl-coumarins
with carboxylic acids. Such carboxylic acids can be further derivatized into
N-hydroxysuccinimidyl (NHS) esters by reaction with TSTU and reacted with amines to
generate analogs with pendant amide groups.
[0108] Figure 37 includes a general synthesis scheme for the preparation of exemplary azetidinyl-acridines.
Reaction of diaminoacridines with acid at elevated temperature can provide dihydroxyacridines,
which can then be reacted with trifluoromethanesulfonic anhydride to access acridine
ditriflates. Acridine ditriflates can then be subjected to Buchwald-Hartwig palladium-catalyzed
C-N cross-coupling with azetidines to prepare azetidinyl-acridines.
[0109] Figure 38 includes a general synthesis scheme for the preparation of exemplary azetidinyl-oxazines.
The ditriflates of phenoxazines can be prepared by reaction of dihydroxyphenoxazines
with trifluoromethanesulfonic anhydride. The ditriflates can be subjected to Buchwald-Hartwig
palladium-catalyzed C-N cross-coupling with azetidines and subsequently oxidized with
DDQ to access azetidinyl-oxazines.
[0110] Those of ordinary skill will recognize that the methods and schemes described herein
are provided for illustrative purposes only and are not intended to limit the scope
of the reactions or reaction sequences useful for synthesizing embodiments of the
presently-disclosed compounds.
EXAMPLES
[0111] The presently-disclosed subject matter is further illustrated by the following specific
but non-limiting examples. The following examples may include compilations of data
that are representative of data gathered at various times during the course of development
and experimentation related to the present invention.
Example 1
[0112] This Example characterizes a structural modification to improve the brightness and
photostability of rhodamine dyes, and specifically a novel azetidinyl auxochrome that
elicited an increase in quantum efficiency relative to the parent dye. The facile
synthesis of the azetidinyl rhodamine dye preserved the spectral properties, cell
permeability, and utility of parent dye. Computational experiments were performed
using the commercial software package Spartan'10 (version 1.1.0, Wavefunction).
[0113] The simplest known rhodamine fluorophore, rhodamine 110 (
Table 2), exhibits an absorption maximum in the blue (
λmax = 497 nm) with a high extinction coefficient (ε = 7.6 × 10
4 M
-1cm
-1), emission in the green (
λem = 520 nm), and a high quantum yield (Φ = 0.88). Alkylation of the rhodamine elicits
a bathochromic shift in absorption and fluorescence emission wavelengths. For example,
tetramethylrhodamine (TMR) shows
λmax/
λem = 548/572 nm and ε = 7.8 × 10
4 M
-1cm
-1 (
Table 2). This shift in spectral properties is accompanied by a significant decrease in quantum
yield, with TMR showing Φ = 0.41. Both of these dyes are used in commercial self-labeling
tag substrates and can be used to label intracellular and extracellular proteins in
living cells.
[0114] The lower quantum efficiency of
N,N,N',N'-tetraalkylrhodamines such as TMR can be explained by an energetically favorable twisted
internal charge transfer (TICT) state (
Figure 1). After excitation, electron transfer from the nitrogen atom to the xanthene ring
results in a pyramidyl nitrogen and a twisted C
aryl-N bond. This TICT state rapidly relaxes without emission of a photon and is a major
path of nonradiative decay in rhodamine dyes. This diradical species may also undergo
irreversible reactions leading to bleaching of the fluorophore. Thus, rhodamine derivatives
where TICT is disfavored should exhibit increased quantum efficiency, longer fluorescence
lifetimes, and higher photostability.
[0115] Using standard
ab initio Hartree-Fock methods to estimate equilibrium geometry, and omitting the
ortho-carboxyl group from the structures to prevent cyclization to the closed lactone form
during energy minimization, the structures shown in
Table 2 were analyzed for the length of the aryl carbon-nitrogen bond (C
aryl-N) and the minimum distance between hydrogen substituents
ortho and alpha to the nitrogen. These values are parameters in the propensity of the molecule
to undergo TICT. A shorter C
aryl-N value signifies increased double-bond character and lower tendency to adopt a twisted
conformation. Likewise, a larger H
o-H
α value indicates less steric clash between substituents and lower predisposition for
bond rotation.
[0116] The calculated structure of the aziridine derivative contained puckered aziridine
rings with the nitrogens out of the plane of the xanthene system, consistent with
the large ring strain (27 kcal mol
-1) present in the three-membered ring. The other rhodamines minimized to a largely
planar structure encompassing the aniline nitrogens, suggesting these dyes prefer
the extended conjugation found in fluorescent rhodamines. The projected structure
of the azetidinyl-rhodamine (JF
549, Example 7) was surprising given the relatively large ring strain present in azetidine
(estimated at 26 kcal mol
-1), which would be expected to favor pyramidal nitrogen atoms. JF
549 showed the shortest C
aryl-N bond length (1.349 Å) and longest H
o-H
α distance (2.56 Å) of the planar calculated structures. Additionally,
N-arylazetidines exhibit higher IP values compared to
N,N-dialkylanilines (1-phenylazetidine IP = 7.61 eV;
N,N-dimethylaniline IP = 7.37 eV) suggesting a higher energetic penalty for the electron
transfer from the aniline nitrogen to the xanthene ring system to form the TICT state.
These results implied that JF
549 would be less prone to undergo TICT and thus exhibit superior fluorescent properties
to the TMR fluorophore (2).
[0117] The compounds of
Table 2 were then synthesized and evaluated for their fluorescence properties. A facile and
efficient synthesis of rhodamines from fluorescein ditriflates using the Buchwald-Hartwig
cross-coupling was used, and allowed the preparation of compounds from fluorescein.
Relatively high catalyst loading (10%) was required to minimize triflate hydrolysis
and ensure high yields. JF
549 and the larger ring structures were highly colored, polar compounds that were purified
by normal-phase flash chromatography with a strong solvent system (CH
2Cl
2/CH
3OH/NH
3). In contrast, the aziridinyl-rhodamine was a colorless, nonpolar molecule and could
be purified by normal-phase chromatography using weak solvent mixtures (EtOAc/hexanes).
[0118] The photophysical properties of the synthesized compounds the compounds were evaluated
in aqueous solution, comparing them to known rhodamine 110 and tetramethylrhodamine.
The data suggested the ring strain in the aziridine substituents forces the aziridinyl-rhodamine
to adopt the closed lactone form. JF
549 (Example 7) and the larger ring structures showed λ
max and λ
em values similar to TMR with increased ring size causing a slight bathochromic shift
of up to 10 nm. Interestingly, JF
549 and the azepane derivative showed a ~30% higher extinction coefficient than the other
dyes. The incorporation of aniline nitrogens into a simple cyclic system was proposed
to control many of the structural parameters in the formation of the TICT state. The
higher ring strain in smaller azacycles such as the aziridine and azetidine-containing
structures was previously believed to preclude the planar configuration required for
the fluorescent xanthenium structure.
[0119] The λ
max, λ
em, and
ε of the different rhodamine dyes showed modest dependence on substituent ring size,
but the fluorescence lifetime (
τ) and quantum yield (
φ) varied as ring size changed (
Table 2). JF
549 exhibited a high quantum yield value (
φ = 0.88) and long fluorescence lifetime (
τ = 3.8 ns), larger than the values for TMR (
φ = 0.41,
τ = 2.2 ns), and similar to the parent rhodamine 110 (1;
φ = 0.88,
τ = 3.3 ns). JF
549 was also 60% brighter than the pyrrolidine derivative , which showed
φ = 0.74 and
τ = 3.6 ns. The piperidine derivative showed a sharp decrease in fluorescence with
φ = 0.10 and
τ = 0.6 ns; the lifetime values for the pyrrolidine and piperidine derivatives were
consistent with those for similar fluorophores. The azepane derivative gave slightly
higher values of
φ = 0.25 and
τ = 1.62 ns relative to the piperidine derivative.
[0120] The improved brightness of JF
549 under one-photon excitation (
Table 2) extended to two-photon excitation and was brought about by a structural change that
preserved many of the desirable properties of TMR. For example, the absorption and
emission spectra of TMR and JF
549 are superimposable (
Figure 2) and the dyes showed comparable sensitivity to solvent polarity (
Figure 3), suggesting similar cell permeability.
Example 2
[0121] This Example describes procedures performed to evaluate the performance of the dye
JF
549 as a label in cellular imaging. JF
549-HaloTag ligand (Example 21) was synthesized starting from a 6-carboxyfluorescein
derivative. The diacetate derivative of 6-carboxyfluorescein was first protected as
a tert-butyl ester. The acetate groups were saponified with NaOH, and this intermediate
was triflated to give 6-
tert-carboxyfluorescein ditriflate in 69% yield over two steps. Cross-coupling with azetidine
gave the rhodamine, which was deprotected to yield the carboxylic acid. Treatment
of the carboxylic acid with DSC followed by reaction with HaloTag(O2)amine yielded
JF
549-HaloTag ligand (Example 21). This molecule was a direct analog of the commercial
TMR-based HaloTag ligand. Notably, rhodamine dyes exist in equilibrium between an
"open," zwitterionic, quinoid form and a "closed," lipophilic, lactone form. This
dynamic amphipathicity makes net neutral rhodamines such as rhodamine 110, TMR, and
JF
549 useful ligands for live-cell labeling technologies, since the dyes efficiently traverse
the cellular membrane without detergents or chemical masking groups and excess ligand
can be rapidly washed away.
[0122] The labeling kinetics of TMR and JF
549 HaloTag ligands were compared with a novel Cy3 HaloTag ligand while measuring the
brightness and photon yield of the resulting conjugates. The JF
549 ligand showed comparable labeling kinetics to the TMR ligand and increased brightness
relative to the other dyes
in vitro. Incubation of live cells expressing a HaloTag-histone 2B (H2B) fusion with the JF
549 ligand resulted in bright nuclear labeling (
Figure 4) and low cytoplasmic background, demonstrating that the JF
549-HaloTag ligand efficiently crossed the membrane of live cells and selectively labeled
the HaloTag protein.
[0123] Incubation of the JF
549 and TMR ligands using low amounts of ligand (< 50 nM) allowed imaging of single molecules
and evaluation of fluorophore brightness (photons/s) and photostability (
i.e., tracklength, s) of individual molecules of labeled HaloTag-H2B. The JF
549 ligand demonstrated a large increase in both brightness and photostability compared
to TMR ligand (
Figure 5). Proteins labeled with TMR ligand showed average photons/s = 1.1 × 10
4 and a mean track length of 0.72 s. Conjugates of JF
549 ligand emitted nearly twice the number of photons/s (1.9 × 10
4) and lasted about twice as long (average track length = 1.6 s). This improvement
in single molecule brightness extended to direct stochastic optical reconstruction
microscopy (
dSTORM) experiments, where the use of a reducing environment enables the reversible
photoswitching of synthetic fluorophores.
[0124] This resulted in a super-resolution image of H2B using the JF
549 ligand (
Figure 6) or TMR ligand (
Figure 7) with median localization errors (
σ) of 14.1 nm and 17.0 nm, respectively (
Figure 8).
dSTORM could be performed inside living cells using the cellular reducing environment
to elicit photoswitching of the JF
549 label (
Figure 9). Thus, JF
549 performed in this spectral range for HaloTag conjugation
in vitro, in fixed cells, and in live cells.
Example 3
[0125] This Example describes the extension of azetidinyl substitution to other dye scaffolds,
including other red-shifted isologs of rhodamines containing carbon and silicon atoms.
This Example demonstrates that the azetidinyl substitution is generalizable to different
fluorophore scaffolds and can produce increases in brightness relative to the original
parent fluorophore scaffolds.
[0126] The
N,N-dialkyl motif is found in numerous classic fluorophore scaffolds (
Table 3), including coumarins (
e.g., Coumarin 461), acridines (
e.g., Acridine Orange), rhodols, carborhodamines, oxazines (e.g., Oxazine 1), and silarhodamines.
TICT has been proposed as a major contributor to nonradiative decay in these fluorescent
systems, leading to modest quantum efficiencies. As with the rhodamines described
in the previous examples, a Pd-catalyzed cross-coupling approach was used to install
the azetidine motif in these fluorophores, starting from accessible aryl halides or
aryl triflates.
Table 3. Spectroscopic data for embodiments of fluorophore scaffolds having the
N,N-dialkyl (i.e., dimethylamino or diethylamino) moiety(ies) replaced with azetidine
rings.
Parent structure |
Substitution |
λmax (nm) |
ε (M-1 cm-1) |
λem (nm) |
Φ |

|

|
372 |
18,000 |
470 |
0.19 |

|
354 |
15,000 |
467 |
0.96 |

|

|
410 |
35,000 |
471 |
0.03 |

|
387 |
24,000 |
470 |
0.84 |

|

|
493 |
50,000 |
528 |
0.21 |

|
492 |
47,000 |
531 |
0.52 |

|

|
518 |
60,000 |
546 |
0.21 |

|
519 |
59,000 |
546 |
0.85 |

|

|
606 |
121,000 |
626 |
0.52 |

|
608 |
99,000 |
631 |
0.67 |

|

|
655 |
111,000 |
669 |
0.07 |

|
647 |
99,000 |
661 |
0.24 |

|

|
643 |
141.000a |
662 |
0.41 |

|
646 |
152.000a |
664 |
0.54 |
All measurements were taken in 10 mM HEPES pH 7.3 unless otherwise noted.
aExtinction coefficient measured in ethanol containing 0.1% v/v trifluoroacetic acid |
[0127] In all cases the azetidine substitution imparted increases in quantum yield without
substantial deleterious effects on other spectral properties (
Table 3). Coumarin 461 exhibited
λmax/
λem = 372 nm/470 nm,
ε = 1.8 × 10
4 M
-1cm
-1, and a modest
φ = 0.19 in aqueous buffer. The azetidine substituted compound (Example 49) showed
a five-fold increase in quantum yield (
φ = 0.96) along with an 18-nm hypsochromic shift in absorbance maxima (
λmax = 354 nm). The emission spectrum and extinction coefficient of the azetidine substituted
compound (
λmax = 467 nm,
ε = 1.5 × 10
4 M
-1cm
-1) were similar to the parent coumarin dye. 7-(Diethylamino)coumarin-3-carboxylic acid
(DEAC) displayed
λma/
λem = 410 nm/471 nm, ε = 3.5 × 10
4 M
-1cm
-1, but a low quantum yield (φ = 0.03). The azetidine -substituted compound (Example
51) showed a shorter absorption maximum (
λmax = 387 nm), a smaller extinction coefficient (
ε = 2.4 × 10
4 M
-1cm
-1), and an emission maxima of
λem = 470 nm, where the azetidine substitution increased the quantum yield by almost
30-fold (
φ = 0.84).
[0128] Next, acridine and rhodol fluorophore scaffolds were modified. The classic fluorophore
Acridine Orange gave
φ = 0.21 when measured in aqueous solution, whereas the azetidine -substituted compound
(Example 60) was 2.5-fold brighter with
φ = 0.52. Other spectral properties of the two acridines were similar. The dimethyl
rhodol showed
λmax/
λem = 518 nm/546 nm,
ε = 6.0 × 10
4 M
-1cm
-1, and
φ = 0.21, and its azetidine-substituted counterpart (Example 59) had similar
λmax,
λem, and
ε values, although replacement of the
N,N-dimethylamino group with an azetidine gave a 4-fold increase in quantum yield (
φ = 0.85).
[0129] Next, with respect to longer-wavelength fluorophores, the carbon-containing analog
of TMR exhibited
λmax/
λem = 606 nm/626 nm,
ε = 1.21 × 10
5 M
-1cm
-1, and
φ = 0.52 in aqueous buffer (
Table 3). The azetidinyl-carborhodamine (Example 40) showed similar absorption and emission
maxima (
λmax/
λem = 608 nm/631 nm) and extinction coefficient (
ε = 9.9 × 10
4 M
-1cm
-1), and a quantum yield
φ = 0.67. The dye Oxazine 1 showed spectral properties in the far red with
λmax/
λem = 655 nm/669 nm,
ε = 1.11 × 10
5 M
-1cm
-1, and a relatively low
φ = 0.07, and azetidine substitution (Example 61) gave a small hypsochromic shift (
λmax/
λem = 647 nm/661 nm), a slightly lower extinction coefficient (
ε = 9.9 × 10
4 M
-1cm
-1), and a 3.4-fold improvement in quantum yield (
φ = 0.24). Finally, the silarhodamine analog of TMR (SiTMR; from
Lukinavič̌̌ius, G.; et al. Nat. Chem. 2013, 5, 132) showed
λmax/
λem = 643 nm/662 nm and
φ = 0.41; the azetidine-substituted compound (Example 30) gave similar absorption and
emission maxima (
λmax/
λem = 646 nm/664 nm) and a higher
φ = 0.54. Since silarhodamines can adopt a colorless form in water, extinction coefficients
were measured in acidic ethanol, finding
ε = 1.41 × 10
5 M
-1cm
-1 for SiTMR and
ε = 1.52 × 10
5 M
-1cm
-1 for the azetidine-substituted compound.
Example 4
[0130] This Example describes cellular imaging using an azetidinyl-silarhodamine. Compounds
based on SiTMR are efficient labels for the SnapTag, HaloTag, and other proteins inside
live cells. The azetidinyl-silarhodamine (Example 30) exhibits superior brightness
(
ε ×
φ,
Table 3) relative to the non-azetidinyl parent compound SiTMR. The azetidinyl-silarhodamine
embodiment described in this Example displays a
λmax = 646, and is referred to herein as "JF
646."
[0131] To compare these two dyes directly in cellular imaging experiments, the HaloTag ligands
of the azetidinyl-silarhodamine (JF646-HaloTag ligand, Example 35) and SiTMR (
Lukinavič̌̌ius, G.; et al. Nat. Chem. 2013, 5, 132) were synthesized from a novel silafluorescein precursor. Both silarhodamine ligands
were excellent labels for super-resolution
dSTORM imaging of HaloTag-H2B (
Figures 10 and 11), showing median localization errors of 8.4 nm and 9.0 nm, respectively (
Figure 12).
dSTORM was also performed on live cells expressing HaloTag-tubulin and labeled with
JF
646 ligand. High photon yields and low background were observed with this label, giving
a median σ = 7.1 nm
(Figures 13 to 15).
[0132] The chromogenicity of the Halotag ligands was compared upon reaction with purified
protein and in live-cell imaging experiments. The SiTMR ligand showed an enhancement
of 6.8-fold upon reaction with excess HaloTag protein in buffer
(Figure 16). The azetidinyl-silarhodamine-HaloTag ligand showed lower background, leading to a
larger, 21-fold increase in absorbance under the same conditions
(Figure 17).
[0133] Next, "no wash" imaging experiments were performed using cells expressing the HaloTag-H2B
fusion. Incubation with either ligand (100 nM) followed directly by wide-field imaging
gave brightly labeled nuclei using both the SiTMR ligand
(Figure 18) and the JF
646 ligand
(Figure 19). SiTMR showed extranuclear fluorescence
(Figure 20), whereas the JF
646 ligand exhibited lower nonspecific staining
(Figures 21
and 22). Overall, these results show the known SiTMR ligand can be replaced with the structurally
similar JF
646 ligand to achieve improved localization error in super-resolution imaging and lower
background in conventional fluorescence microscopy.
Example 5
[0134] This Example describes a multiplexed single particle tracking and super resolution
imaging process performed in the same cell. Given the spectral separation between
JF
549 and JF
646, two distinct protein species were imaged at the single-molecule level in the same
living cell.
[0135] To achieve orthogonal labeling, the SnapTag ligand of JF
549 was prepared. HaloTag-H2B and a fusion of the SnapTag enzyme and the Tet repressor
protein (SnapTag-TetR) were coexpressed and labeled with, respectively, JF
646-HaloTag ligand (Example 35) and JF
549-SnapTag ligand (Example 22). The trajectories of individual JF
549-labeled TetR proteins were imaged, and a rapid live-cell
dSTORM experiment of the JF
646-H2B conjugate was subsequently performed
(Figure 23). This two-color procedure revealed the respective partitions of fast- and slow-diffusing
DNA-binding protein in relation to the chromatin structure of the nucleus
(Figure 24). Histograms of the diffusion coefficient of both the H2B-colocalized and the non-colocalized
TetR trajectories were then plotted and showed TetR colocalized with H2B to a greater
extent than with non-colocalized positions
(Figure 25).
[0136] Thus, the present compounds may be used in multicolor experiments in living cells,
where several components involved in a biological process can be tracked and localized
with molecular precision within the same cell.
Example 6
[0137] In this Example, an azetidinyl-coumarin label was used for cellular imaging. The
performance of a commercial SnapTag ligand (i.e., Snap Cell 430) was compared to a
novel azetidinyl derivative (Example 52), which was synthesized from a 7-azetidinyl-coumarin-3-carboxylic
acid. Under identical transient transfection, labeling, and imaging conditions, H2B-SnapTag-expressing
cells were stained with the red fluorescent nuclear stain DRAQ5 and either Snap Cell
430 or the azetidinyl ligand. Using the DRAQ5 staining as a spatial reference
(Figures 26 and 27) the intensity of individual nuclei labeled by either SnapTag ligand was measured.
Cells incubated with Snap Cell 430 ligand showed low fluorescence intensity
(Figure 28), whereas cells labeled with the azetidinyl-coumarin SnapTag ligand exhibited brighter
nuclear labeling
(Figure 29). Quantification of nuclear intensity showed the cells labeled with azetidine had median
values that were five-fold higher than cells labeled with the commercial compound
(Figure 30).
Examples 7-61
[0138] The following examples describe specific embodiments of the compounds described herein,
and illustrates the flexibility of the present azetidine approach for making dyes.
We reasoned that we could further tune the physicochemical properties of JF
549 by exploring different substitution patterns at the 3-position of the azetidine.
For instance, the substituted azetidinyl-rhodamines shown in
Table 1 had relatively high
ε values and quantum yield values.
[0139] Fluorescent and fluorogenic molecules for spectroscopy were prepared as stock solutions
in DMSO and diluted such that the DMSO concentration did not exceed 1% v/v. Phosphate
buffered saline (PBS) was at pH 7.4 unless otherwise noted.
[0140] Spectroscopy was performed using 1-cm path length, 3.5-mL quartz cuvettes from Starna
Cells or 1-cm path length, 1.0-mL quartz microcuvettes from Hellma. All measurements
were taken at ambient temperature (22 ± 2 °C) in 10 mM HEPES, pH 7.3 buffer unless
otherwise noted. Absorption spectra were recorded on a Cary Model 100 spectrometer
(Varian); reported values for extinction coefficients (
ε) are averages (n = 3). Fluorescence spectra were recorded on a Cary Eclipse fluorometer
(Varian). Normalized spectra are shown for clarity.
[0141] All reported quantum yield values were measured in under identical conditions using
a Quantaurus-QY spectrometer (C11374, Hamamatsu). Measurements were carried out using
dilute samples (
A < 0.1) and self-absorption corrections were performed using the instrument software.
Reported values are averages (n = 3).
[0142] Dioxane-H
2O titrations were performed in spectral grade dioxane (Sigma-Aldrich) and milliQ H
2O. The solvent mixtures contained 0.01% v/v triethylamine to ensure the rhodamine
dyes were in the zwitterionic form. The absorbance values at λ
max were measured on 5 µM samples (n = 2) using a quartz 96-well microplate (Hellma)
and a FlexStation3 microplate reader (Molecular Devices).
[0143] For fluorescence lifetime measurements, a pulse picker (Model 350-160, ConOptics)
was placed in the laser beam to reduce the pulse frequency from 80 MHz to 20 MHz.
Samples (2 µM dye diluted in 50 mM HEPES, pH 7.2, H
2O, or CH
3OH) were excited at 830 nm laser wavelength and 6 mW laser power. The emitted light
was collected by the fast-timing APD and fed to the single-photon counting board (TimeHarp200;
PicoQuant). Timing pulses were obtained from a PIN diode (DET01CFC; ThorLabs) monitoring
the 20 MHz pulse train. The temporal impulse response of the system was determined
by second harmonic generation of laser pulses using a thin nonlinear crystal in place
of a dye sample. The lifetime decay data was fit to a single exponential decay function
using a custom MATLAB program. Lifetime value of the reference fluorescein dye measured
using this system was 4.025 ± 0.015 ns (R
2 = 0.99) compared to a literature value of 4.1 ± 0.1 ns (
Magde, D.; Rojas, G. E.; Seybold, P. G. Photochem. Photobiol. 1999, 70, 737).
[0144] To measure fluorescence of HaloTag ligands upon reaction with HaloTag protein, absorbance
measurements were performed in 1 mL quartz cuvettes. HaloTag protein was used as a
100 µM solution in 75 mM NaCl, 50 mM TRIS·HCl, pH 7.4 with 50% v/v glycerol (TBS-glycerol).
HaloTag ligands of JF
646 and SiTMR (5 µM) were dissolved in 10 mM HEPES, pH 7.3 containing 0.1 mg·mL
-1 CHAPS. An aliquot of HaloTag protein (1.5 equiv) or an equivalent volume of TBS-glycerol
blank was added and the resulting mixture was incubated until consistent absorbance
signal was observed (∼30 min). Additional HaloTag protein did not elicit an increase
in absorbance (not shown). Absorbance scans are averages (n = 2).
[0145] HeLa cells (ATCC) and U2OS cells (ATCC) were cultured in Dulbecco's modified eagle
medium (DMEM; Life Technologies) supplemented with 10% v/v fetal bovine serum (FBS;
Life Technologies), 1 mM GlutaMax (Life Technologies), and 1 mM sodium pyruvate (Sigma)
and maintained at 37 °C in a humidified 5% v/v CO
2 environment. These cell lines undergo regular mycoplasma testing by the Janelia Cell
Culture Facility. Cells were transfected with HaloTag-H2B, HaloTag-tubulin, SnapTag-TetR,
or SnapTag-H2B using an Amaxa Nucleofector (Lonza). Before the imaging experiments,
transfected cells were transferred onto a No.1 coverslip (Warner Instruments) that
was cleaned by Piranha solution (3:1 v/v mixture of concentrated H
2SO
4 and 30% v/v hydrogen peroxide). To label live cells with the HaloTag or SnapTag ligands,
the ligands were added to the growth medium and the samples incubated for 15 min.
Labeling concentrations were typically 100-500 nM for confocal, wide-field, and
dSTORM experiments and 5-50 nM for single-molecule tracking experiments. Cells were
then washed briefly with PBS (1×) and then incubated in DMEM-FBS for an additional
15 min. Before imaging, the cells were washed briefly with PBS (3×) and placed in
fresh DMEM-FBS for imaging. All washes were omitted in the "no wash" experiments.
For nuclear staining, cells were incubated in PBS for 5 min (2×), and then incubated
in PBS containing 5 µM DRAQ5 (Cell Signaling) for 5 min, followed by brief wash with
PBS (1×). During all imaging experiments, cells were maintained at 37 °C in a humidified
5% CO
2 v/v environment supplied by a live-cell incubator (TOKAI HIT).
[0146] Three separate systems were used to acquire microscopic images. Confocal microscopy
was performed using a Zeiss LSM 510 META confocal microscope with a LD C-APOCHROMAT
40×/1.2 W Korr M27 UV-VIS-IR objective. Wide-field microscopy, 2D single-molecule
tracking, and super-resolution imaging experiments were conducted on a Nikon Eclipse
Ti wide field epifluorescence microscope equipped with a 100×, 1.4NA oil-immersion
objective lens (Nikon), a Lumencor light source, a set of lasers (405 nm/100 mW, Coherent
Cube; 561 nm/200 mW, Cobolt Jive; 633 nm/140 mW, Vortran Stradus), controlled by an
Acousto-Optic Tunable Filter (AA Opto-Electronic), two filter wheels (Lambda 10-3;
Sutter Instruments), a perfect focusing system (Nikon), and an EMCCD camera (iXon3,
Andor). Emission filters (FF01 593/40 or FF01 676/37; Semrock) were placed in front
of the cameras for JF
549 and JF
646 emission. A multi-band mirror (405/488/561/633 BrightLine quad-band bandpass filter,
Semrock) was used to reflect the excitation laser beams into the objective. The microscope,
cameras, and hardware were controlled through the NIS-Elements software (Nikon). Other
live-cell single super-resolution imaging experiments were recorded on a custom-built
three-camera RAMM frame (ASI) microscope using an 1.4NA PLAPON 60× OSC objective (Olympus),
and a 300 mm focal length tube lens (LAO-300.0, Melles Griot), resulting in 100× overall
magnification. Stroboscopic 405 nm excitation of the Stradus 405-100 laser (Vortran)
was achieved using a NI-DAQ-USB-6363 acquisition board (National Instruments), which
also controlled the 637 nm laser emission from a Stradus 637-140 laser (Vortran).
A 2mm-thick quad-band dichroic (ZT 405/488/561/640rpx, Chroma), and a band-pass emission
filter (FF01-731/137-25, Semrock) filtered the emitted light. Fluorescence was detected
with a back-illuminated EMCCD camera (Andor Technology, Ixon Ultra DU-897U-CS0-EXF,
17 MHz EM amplifier), which was controlled through Micro-Manager (1.4.17).
[0147] For live-cell
dSTORM imaging the cells were labeled, washed, and imaged directly in DMEM-FBS. For
fixed cell preparations, cells were labeled, washed, and fixed in 4% paraformaldehyde
(Electron Microscopy Sciences) in PBS buffer (pH = 7.5). The cells were imaged in
a sealed cell chamber (Life Technologies) containing nitrogen-degassed redox buffer
consisting of PBS supplemented with 50 mM mercaptoethylamine (Sigma-Aldrich), 10%
w/v glucose, 0.5 mg/mL glucose oxidase (Sigma-Aldrich), and 28400 U/mL catalase (Sigma-Aldrich).
Before imaging, JF
549 could be efficiently "shelved" in a dark state upon illumination with 2 kW·cm
-2 of excitation light (561 nm), and then activated back to a fluorescent state by blue
light (405 nm) with low intensity (∼20·Wcm
-2). JF
646 fluorophores were converted into a predominately dark state using continuous illumination
of 637 nm excitation light at 14 kW·cm
-2, after which individual rapidly blinking molecules of JF
646 fluorophores were observed. These experiments were conducted on the two wide-field
microscope systems described above: the Nikon Eclipse Ti epifluorescence microscope
and the custom-built three-camera microscope with an ASI RAMM frame.
[0148] The spot localization (x,y) was obtained based on the multiple-target tracing (MTT)
algorithm (
Serge, A.; Bertaux, N.; Rigneault, H.; Marguet, D. Nature Protocol Exchange 2008,
doi:10.1038/nprot.2008.1128;
Serge, A.; Bertaux, N.; Rigneault, H.; Marguet, D. Nat. Methods 2008, 5, 687) using a custom MATLAB program. For each frame, the PSF of individual fluorophores
was fitted into a two-dimensional Gaussian distribution. Integrated fluorescence intensities
were calculated and converted to photon counts using analysis routines written in
IGOR Pro version 6.34A. Localization errors were calculated using Equation 6 in Mortensen
et al. (
Mortensen, K. I.; Churchman, L. S.; Spudich, J. A.; Flyvbjerg, H. Nat. Methods 2010,
7, 377). Super-resolution images were rendered using the software package Localizer by Dedecker
et al. (
Dedecker, P.; Duwé, S.; Neely, R. K.; Zhang, J. J. Biomed. Opt. 2012, 17, 126008) running from Igor Pro v. 3.34A, which superimposes the position coordinates of detected
spots as Gaussian masks using the fitted intensity values as amplitudes and the localization
errors as the widths. The
dSTORM data for experiments comparing two different fluorophore ligands was recorded
on the same day under identical illumination conditions.
[0149] The two-color single-molecule experiments were recorded on the Nikon Eclipse Ti wide
field epifluorescence microscope. We first performed a 2D single molecule tracking
of SnapTag-TetR-JF
549 using a 561-nm laser of excitation intensity ∼1 kW cm
-2 at a frame rate of 100 Hz. Immediately after the completion of the single-particle
tracking experiment, we then imaged HaloTag-H2B-JF
646 under the
dSTORM mode as described above. Transmission images were taken before and after the
tracking-
dSTORM experiments and a cross-correlation algorithm was employed to calculate the
image drift (
Guizar-Sicairos, M.; Thurman, S. T.; Fienup, J. R. Opt. Lett. 2008, 33, 156). Tracking analysis of TetR was performed using the commercial tracking software
DiaTrack (v. 3.03, Semasopht), which identifies and fits the intensity spots of fluorescent
particles with 2D Gaussian functions matched to the experimentally determined point-spread
function. The diffusion map was created using tracking routines written in IGOR Pro
6.34A, calculating the local apparent diffusion of TetR mobility evaluated on a 20
nm × 20 nm x-y grid from the mean square displacements over a timescale of 10 milliseconds.
Whenever two or more separate displacements originating within 80 nm of a given grid
node were found, a local apparent diffusion coefficient was calculated and plotted.
H2B clusters were then selected as the 500 brightest spots in the super-resolved image.
From this analysis, a histogram of apparent diffusion coefficients for all trajectories
that dwelled within 320 nm of a H2B cluster for at least 10 milliseconds was generated.
Histograms of the diffusion coefficient of both the H2B-colocalized and the non-colocalized
TetR trajectories were then plotted.
Example 7. 2-(3,6-Di(azetidin-1-yl)xanthylium-9-yl)benzoate
[0150]

[0151] A vial was charged with fluorescein ditriflate (75 mg, 126 µmol; from
Grimm, J. B.; Lavis, L. D. Org. Lett. 2011, 13, 6354), Pd
2dba
3 (11.5 mg, 12.6 µmol, 0.1 eq), XPhos (18.0 mg, 37.7 µmol, 0.3 eq), and Cs
2CO
3 (115 mg, 352 µmol, 2.8 eq). The vial was sealed and evacuated/backfilled with nitrogen
(3×). Dioxane (1 mL) was added, and the reaction was flushed again with nitrogen (3×).
Following the addition of azetidine (20.3 µL, 302 µmol, 2.4 eq), the reaction was
stirred at 100 °C for 18 h. It was then cooled to room temperature, diluted with MeOH,
deposited onto Celite, and concentrated to dryness. Purification by silica gel chromatography
(0-10% MeOH (2 M NH
3)/CH
2Cl
2, linear gradient; dry load with Celite) afforded the title compound (49 mg, 95%)
as a purple solid.
1H NMR (CDCl
3, 400 MHz) δ 8.03 - 7.96 (m, 1H), 7.63 (td,
J = 7.4, 1.3 Hz, 1H), 7.58 (td,
J = 7.4, 1.1 Hz, 1H), 7.20 - 7.13 (m, 1H), 6.56 (d,
J = 8.6 Hz, 2H), 6.20 (d,
J = 2.3 Hz, 2H), 6.09 (dd,
J = 8.6, 2.3 Hz, 2H), 3.91 (t,
J = 7.3 Hz, 8H), 2.37 (p,
J= 7.2 Hz, 4H);
13C NMR (CDCl
3, 101 MHz) δ 169.9 (C), 153.7 (C), 153.1 (C), 152.9 (C), 134.6 (CH), 129.4 (CH), 129.0
(CH), 127.8 (C), 125.0 (CH), 124.3 (CH), 107.9 (C), 107.8 (CH), 97.7 (CH), 52.2 (CH
2), 16.8 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
26H
23N
2O
3 [M+H]
+ 411.1703, found 411.1714.
Example 8. 2-(3,6-Bis(3,3-dimethylazetidin-1-yl)xanthylium-9-yl)benzoate
[0152]

[0153] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and 3,3-dimethylazetidine hydrochloride (86%, purple solid).
1H NMR (MeOD, 400 MHz) δ 8.11 - 8.06 (m, 1H), 7.67 - 7.57 (m, 2H), 7.23 - 7.20 (m,
1H), 7.19 (d,
J = 9.2 Hz, 2H), 6.56 (dd,
J = 9.1, 2.2 Hz, 2H), 6.49 (d,
J = 2.2 Hz, 2H), 3.92 (s, 8H), 1.39 (s, 12H);
13C NMR (MeOD, 101 MHz) δ 173.1 (C), 160.2 (C), 158.7 (C), 158.2 (C), 140.8 (C), 134.9
(C), 132.9 (CH), 130.82 (CH), 130.77 (CH), 130.74 (CH), 130.2 (CH), 115.0 (C), 113.1
(CH), 95.5 (CH), 64.4 (CH
2), 33.0 (C), 27.1 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
30H
31N
2O
3 [M+H]
+ 467.2329, found 467.2341.
Example 9. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)xanthylium-9-yl)benzoate
[0154]

[0155] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and 3,3-difluoroazetidine hydrochloride (91%, pink solid).
1H NMR (CDCl
3, 400 MHz) δ 8.03 - 7.99 (m, 1H), 7.66 (td,
J = 7.4, 1.3 Hz, 1H), 7.60 (td,
J = 7.4, 1.1 Hz, 1H), 7.17 - 7.14 (m, 1H), 6.64 (d,
J = 8.6 Hz, 2H), 6.30 (d,
J = 2.4 Hz, 2H), 6.17 (dd,
J = 8.6, 2.4 Hz, 2H), 4.25 (t,
3JHF = 11.7 Hz, 8H);
19F NMR (CDCl
3, 376 MHz) 8 -100.05 (p,
3JFH = 11.8 Hz);
13C NMR (CDCl
3, 101 MHz) δ 169.6 (C), 153.3 (C), 152.6 (C), 151.3 (t,
4JCF = 2.9 Hz, C), 135.0 (CH), 129.7 (CH), 129.3 (CH), 127.2 (C), 125.1 (CH), 124.0 (CH),
115.8 (t,
1JCF = 274.6 Hz, CF
2), 109.7 (C), 108.8 (CH), 99.4 (CH), 83.9 (C), 63.4 (t,
2JCF = 26.3 Hz, CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 525 nm); HRMS (ESI) calcd for C
26H
19F
4N
2O
3 [M+H]
+ 483.1326, found 483.1336.
Example 10. 2-(3,6-Bis(3-fluoroazetidin-1-yl)xanthylium-9-yl)benzoate
[0156]

[0157] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and 3-fluoroazetidine hydrochloride (89%, pink solid).
1H NMR (DMSO-d
6, 400 MHz) δ 8.01 - 7.95 (m, 1H), 7.78 (td,
J = 7.5, 1.2 Hz, 1H), 7.71 (td,
J = 7.5, 0.9 Hz, 1H), 7.25 - 7.20 (m, 1H), 6.52 (d,
J = 8.6 Hz, 2H), 6.33 (d,
J = 2.3 Hz, 2H), 6.24 (dd,
J = 8.6, 2.3 Hz, 2H), 5.49 (dtt,
2JHF = 57.6 Hz,
J = 6.0, 3.1 Hz, 2H), 4.26 - 4.13 (m, 4H), 4.00 - 3.88 (m, 4H);
19F NMR (DMSO-d
6, 376 MHz) δ - 178.95 (dtt,
JFH = 57.4, 24.2, 20.9 Hz);
13C NMR (DMSO-d
6, 101 MHz) δ 168.7 (C), 152.54 (d,
4JCF = 1.3 Hz, C), 152.47 (C), 151.8 (C), 135.4 (CH), 129.9 (CH), 128.6 (CH), 126.4 (C),
124.5 (CH), 123.9 (CH), 108.6 (CH), 107.8 (C), 98.0 (CH), 83.8 (C), 83.3 (d,
1JCF = 200.3 Hz, CFH), 59.2 (d,
2JCF = 23.7 Hz, CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
26H
21F
2N
2O
3 [M+H]
+ 447.1515, found 447.1525.
Example 11. 2-(3,6-Bis(3-methoxyazetidin-1-yl)xanthylium-9-yl)benzoate
[0158]

[0159] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and 3-methoxyazetidine hydrochloride (83%, purple solid).
1H NMR (DMSO-d
6, 400 MHz) δ 8.00 - 7.94 (m, 1H), 7.77 (td,
J = 7.5, 1.2 Hz, 1H), 7.70 (td,
J = 7.5, 0.9 Hz, 1H), 7.25 - 7.20 (m, 1H), 6.48 (d,
J = 8.6 Hz, 2H), 6.26 (d,
J = 2.3 Hz, 2H), 6.19 (dd,
J = 8.6, 2.3 Hz, 2H), 4.32 (tt,
J = 6.2, 4.2 Hz, 2H), 4.07 (dd,
J= 8.0, 6.6 Hz, 4H), 3.66 (dd,
J= 8.4, 4.1 Hz, 4H), 3.24 (s, 6H);
13C NMR (DMSO-d
6, 101 MHz) δ 168.7 (C), 152.8 (C), 152.5 (C), 151.9 (C), 135.4 (CH), 129.9 (CH), 128.5
(CH), 126.5 (C), 124.5 (CH), 123.9 (CH), 108.2 (CH), 107.2 (C), 97.5 (CH), 84.1 (C),
69.2 (CH), 58.3 (CH
2), 55.4 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
28H
27N
2O
5 [M+H]
+ 471.1914, found 471.1926.
Example 12. 2-(3,6-Bis(3-cyanoazetidin-1-yl)xanthylium-9-yl)benzoate
[0160]

[0161] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and 3-azetidinecarbonitrile hydrochloride (85%, magenta solid).
1H NMR (CDCl
3, 400 MHz) δ 8.03 - 7.98 (m, 1H), 7.66 (td,
J = 7.4, 1.3 Hz, 1H), 7.60 (td,
J = 7.4, 1.1 Hz, 1H), 7.17 - 7.13 (m, 1H), 6.62 (d,
J = 8.6 Hz, 2H), 6.25 (d,
J = 2.3 Hz, 2H), 6.12 (dd,
J = 8.6, 2.4 Hz, 2H), 4.25 - 4.18 (m, 4H), 4.15 - 4.08 (m, 4H), 3.60 (tt,
J = 8.5, 6.2 Hz, 2H);
13C NMR (CDCl
3, 101 MHz) δ 169.6 (C), 153.2 (C), 152.5 (C), 151.9 (C), 135.0 (CH), 129.7 (CH), 129.3
(CH), 127.1 (C), 125.1 (CH), 124.0 (CH), 119.7 (C), 109.7 (C), 108.1 (CH), 98.7 (CH),
83.9 (C), 55.2 (CH
2), 18.4 (CH); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL
injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
28H
21N
4O
3 [M+H]
+ 461.1608, found 461.1628.
Example 13. 2-(3,6-Bis(3-(dimethylamino)azetidin-1-yl)xanthylium-9-yl)benzoate
[0162]

[0163] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and 3-(dimethylamino)azetidine dihydrochloride (80%, purple
solid).
1H NMR (MeOD, 400 MHz) δ 8.10 - 8.05 (m, 1H), 7.69 - 7.60 (m, 2H), 7.24 - 7.19 (m,
1H), 7.12 (d,
J = 9.0 Hz, 2H), 6.56 (dd,
J = 9.0, 2.2 Hz, 2H), 6.53 (d,
J = 2.2 Hz, 2H), 4.31 - 4.22 (m, 4H), 4.01 (dd,
J = 10.5, 5.1 Hz, 4H), 3.39 (tt,
J = 7.0, 5.1 Hz, 2H), 2.27 (s, 12H);
13C NMR (MeOD, 101 MHz) δ 172.8 (C), 157.9 (C), 157.1 (C), 147.7 (C), 138.7 (C), 138.4
(C), 132.5 (CH), 131.8 (CH), 130.8 (CH), 129.9 (CH), 129.3 (CH), 114.1 (C), 112.4
(CH), 96.3 (CH), 57.0 (CH), 56.6 (CH
2), 42.0 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
30H
33N
4O
3 [M+H]
+ 497.2547, found 497.2561.
Example 14. 2-(3,6-Bis(3-(methoxycarbonyl)azetidin-1-yl)xanthylium-9-yl)benzoate
[0164]

[0165] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and methyl azetidine-3-carboxylate hydrochloride (79%, purple
solid).
1H NMR (MeOD, 400 MHz) δ 8.09 - 8.03 (m, 1H), 7.69 - 7.62 (m, 2H), 7.24 - 7.17 (m,
1H), 7.02 (d,
J = 8.9 Hz, 2H), 6.48 (dd,
J = 8.9, 2.2 Hz, 2H), 6.45 (d,
J = 2.1 Hz, 2H), 4.34 (t,
J = 9.0 Hz, 4H), 4.25 (dd,
J = 9.0, 5.9 Hz, 4H), 3.77 (s, 6H), 3.71 (tt,
J = 8.9, 5.9 Hz, 2H);
13C NMR (MeOD, 101 MHz) δ 174.4 (C), 172.6 (C), 157.0 (C), 156.5 (C), 141.6 (C), 136.7
(C), 135.8 (C), 132.7 (CH), 131.9 (CH), 130.9 (CH), 129.0 (CH), 128.5 (CH), 113.3
(C), 111.7 (CH), 96.8 (CH), 55.2 (CH
2), 52.9 (CH
3), 34.0 (CH); HRMS (ESI) calcd for C
30H
27N
2O
7 [M+H]
+ 527.1813, found 527.1823.
Example 15. 2-(3,6-Bis(3-(2-methoxy-2-oxoethyl)azetidin-1-yl)xanthylium-9-yl)benzoate
[0166]

[0167] The procedure described for Example 7 was used to prepare the title compound from
fluorescein ditriflate and methyl 3-azetidineacetate trifluoroacetate (67%, purple
solid).
1H NMR (MeOD, 400 MHz) δ 8.10 - 8.05 (m, 1H), 7.67 - 7.57 (m, 2H), 7.21 - 7.18 (m,
1H), 7.16 (d,
J= 9.1 Hz, 2H), 6.54 (dd,
J= 9.1, 2.2 Hz, 2H), 6.48 (d,
J= 2.2 Hz, 2H), 4.41 - 4.32 (m, 4H), 3.97 - 3.88 (m, 4H), 3.69 (s, 6H), 3.26 - 3.13
(m, 2H), 2.80 (d,
J = 7.7 Hz, 4H);
13C NMR (MeOD, 101 MHz) δ 173.7 (C), 173.0 (C), 158.3 (C), 157.5 (C), 154.9 (C), 140.1
(C), 136.3 (C), 132.7 (CH), 131.1 (CH), 130.8 (CH), 130.4 (CH), 129.8 (CH), 114.5
(C), 112.7 (CH), 95.7 (CH), 57.5 (CH
2), 52.2 (CH
3), 38.5 (CH
2), 27.3 (CH); HRMS (ESI) calcd for C
32H
31N
2O
7 [M+H]
+ 555.2126, found 555.2132.
Example 16. 2-(3,6-Bis(3-carboxyazetidin-1-yl)xanthylium-9-yl)benzoate
[0168]

[0169] 2-(3,6-Bis(3-(methoxycarbonyl)azetidin-1-yl)xanthylium-9-yl)benzoate (Example 14;
40 mg, 76.0 µmol) was dissolved in MeOH (2.5 mL), and 1 M NaOH (304 µL, 304 µmol,
4 eq) was added. After stirring the reaction at room temperature for 18 h, it was
acidified with 1 M HCl (350 µL) and directly purified by reverse phase HPLC (10-50%
MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) to provide 28 mg (60%, TFA
salt) of the title compound as a red-purple solid.
1H NMR (MeOD, 400 MHz) δ 8.36 - 8.30 (m, 1H), 7.84 (td,
J = 7.5, 1.6 Hz, 1H), 7.79 (td,
J = 7.6, 1.5 Hz, 1H), 7.40 - 7.36 (m, 1H), 7.12 (d,
J = 9.2 Hz, 2H), 6.66 (dd,
J = 9.2, 2.2 Hz, 2H), 6.61 (d,
J = 2.2 Hz, 2H), 4.48 (t,
J = 9.6 Hz, 4H), 4.39 (dd,
J = 9.9, 5.9 Hz, 4H), 3.72 (tt,
J = 9.0, 5.8 Hz, 2H);
13C NMR (MeOD, 101 MHz) δ 175.2 (C), 168.0 (C), 162.4 (C), 158.9 (C), 157.9 (C), 135.3
(C), 133.9 (CH), 132.6 (CH), 132.5 (CH), 131.5 (CH), 131.4 (CH), 115.4 (C), 113.8
(CH), 95.6 (CH), 55.3 (CH
2), 33.9 (CH); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL
injection; 10-75% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
28H
23N
2O
7 [M+H]
+ 499.1500, found 499.1507.
Example 17. 2-(3,6-Bis(3-(carboxymethyl)azetidin-1-yl)xanthylium-9-yl)benzoate
[0170]

[0171] The procedure described for Example 16 was used to prepare the title compound from
Example 15 (80%, red-purple solid, TFA salt).
1H NMR (MeOD, 400 MHz) δ 8.35 - 8.30 (m, 1H), 7.83 (td,
J= 7.5, 1.5 Hz, 1H), 7.78 (td,
J = 7.6, 1.5 Hz, 1H), 7.40 - 7.35 (m, 1H), 7.07 (d,
J = 9.2 Hz, 2H), 6.62 (dd,
J= 9.2, 2.2 Hz, 2H), 6.56 (d,
J = 2.2 Hz, 2H), 4.43 (t,
J = 9.6 Hz, 4H), 4.05 - 3.96 (m, 4H), 3.28 - 3.16 (m, 2H), 2.78 (d,
J = 7.7 Hz, 4H);
13C NMR (MeOD, 101 MHz) δ 175.1 (C), 167.9 (C), 161.7 (C), 158.8 (C), 158.0 (C), 135.4
(C), 133.8 (CH), 132.5 (CH), 132.3 (CH), 131.41 (CH), 131.40 (CH), 115.1 (C), 113.7
(CH), 95.3 (CH), 57.6 (CH
2), 38.5 (CH
2), 27.3 (CH); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL
injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
30H
27N
2O
7 [M+H]
+ 527.1813, found 527.1815.
Example 18. 4-(tert-Butoxycarbonyl)-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate
[0172]

[0173] Step 1: A suspension of 6-carboxyfluorescein diacetate (1.39 g, 3.02 mmol) in toluene (6
mL) was heated to 80 °C, and
N,N-dimethylformamide di-
tert-butyl acetal (4.34 mL, 18.1 mmol, 6 eq) was added dropwise over 5 min. The reaction
was stirred at 80 °C for 15 min. After cooling the mixture to room temperature, it
was diluted with saturated NaHCO
3 and extracted with CH
2Cl
2 (2×). The combined organic extracts were dried (MgSO
4), filtered, and evaporated. Flash chromatography (0-20% EtOAc/hexanes, linear gradient,
with constant 40% v/v CH
2Cl
2) provided 6-(
tert-butoxycarbonyl)-3-oxo-3
H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl diacetate as a colorless solid (971
mg, 62%).
1H NMR (CDCl
3, 400 MHz) δ 8.26 (dd,
J = 8.0, 1.3 Hz, 1H), 8.07 (dd,
J = 8.0, 0.7 Hz, 1H), 7.73 (dd,
J = 1.2, 0.8 Hz, 1H), 7.12 (dd,
J = 2.1, 0.4 Hz, 2H), 6.84
(dd, J= 8.7, 2.1 Hz, 2H), 6.80
(dd, J= 8.7, 0.5 Hz, 2H), 2.32 (s, 6H), 1.56 (s, 9H);
13C NMR (CDCl
3, 101 MHz) δ 168.9 (C), 168.3 (C), 164.0 (C), 152.8 (C), 152.3 (C), 151.7 (C), 138.8
(C), 131.4 (CH), 129.4 (C), 129.1 (CH), 125.2 (CH), 125.1 (CH), 118.0 (CH), 116.0
(C), 110.6 (CH), 83.0 (C), 82.1 (C), 28.2 (CH
3), 21.3 (CH
3); HRMS (ESI) calcd for C
29H
25O
9 [M+H]
+ 517.1493, found 517.1495.
[0174] Step 2: To a solution of the intermediate from Step 1 (910 mg, 1.76 mmol) in 1:1 THF/MeOH
(20 mL) was added 1 M NaOH (4.23 mL, 4.23 mmol, 2.4 eq). The reaction was stirred
at room temperature for 1 h. The resulting red-orange solution was acidified with
1 N HCl (5 mL), diluted with water, and extracted with EtOAc (2×). The organics were
washed with brine, dried (MgSO
4), filtered, and concentrated
in vacuo to provide a red solid. The crude solid was suspended in CH
2Cl
2 (15 mL) and cooled to 0 °C. Pyridine (1.14 mL, 14.1 mmol, 8 eq) and trifluoromethanesulfonic
anhydride (1.19 mL, 7.05 mmol, 4 eq) were added, and the ice bath was removed. The
reaction was stirred at room temperature for 1 h. It was subsequently diluted with
water and extracted with CH
2Cl
2 (2×). The combined organic extracts were dried (MgSO
4), filtered, and evaporated. Silica gel chromatography (0-25% EtOAc/hexanes, linear
gradient) yielded 841 mg (69%) of
tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3
H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate as a colorless solid.
1H NMR (CDCl
3, 400 MHz) δ 8.28 (dd,
J = 8.0, 1.3 Hz, 1H), 8.11 (dd,
J = 8.0, 0.7 Hz, 1H), 7.75 (dd,
J = 1.2, 0.7 Hz, 1H), 7.32 (d,
J = 2.4 Hz, 2H), 7.04 (dd,
J = 8.8, 2.5 Hz, 2H), 6.94 (d,
J = 8.8 Hz, 2H), 1.57 (s, 9H);
19F NMR (CDCl
3, 376 MHz) δ - 73.12 (s);
13C NMR (CDCl
3, 101 MHz) δ 167.7 (C), 163.8 (C), 152.2 (C), 151.5 (C), 150.5 (C), 139.3 (C), 131.9
(CH), 130.1 (CH), 128.8 (C), 125.8 (CH), 124.9 (CH), 118.9 (C), 118.8 (q,
1JCF = 320.9 Hz, CF
3), 118.0 (CH), 111.0 (CH), 83.3 (C), 80.5 (C), 28.2 (CH
3); HRMS (ESI) calcd for C
27H
19F
6O
11S
2 [M+H]
+ 697.0267, found 697.0255.
[0175] Step 3: The procedure described for Example 7 was used to prepare the title compound 4-(
tert-butoxycarbonyl)-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate from the ditriflate
synthesized in Step 2 (86%, dark purple solid).
1H NMR (CDCl
3, 400 MHz) δ 8.19 (dd,
J = 8.0, 1.4 Hz, 1H), 8.02 (dd,
J= 8.0, 0.7 Hz, 1H), 7.73 (dd,
J = 1.3, 0.7 Hz, 1H), 6.55 (d,
J = 8.6 Hz, 2H), 6.21 (d,
J = 2.3 Hz, 2H), 6.09 (dd,
J= 8.6, 2.3 Hz, 2H), 3.92 (t,
J = 7.3 Hz, 8H), 2.38 (p,
J = 7.2 Hz, 4H), 1.54 (s, 9H);
13C NMR (CDCl
3, 101 MHz) δ 169.1 (C), 164.5 (C), 153.8 (C), 153.0 (C), 152.6 (C), 137.9 (C), 131.1
(C), 130.6 (CH), 129.0 (CH), 125.4 (CH), 125.0 (CH), 107.9 (CH), 107.4 (C), 97.6 (CH),
82.4 (C), 52.2 (CH
2), 28.2 (CH
3), 16.8 (CH
2); HRMS (ESI) calcd for C
31H
31N
2O
5 [M+H]
+ 511.2227, found 511.2253.
Example 19. 4-Carboxy-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate
[0176]

[0177] 4-(
tert-Butoxycarbonyl)-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate (Example 18; 70
mg, 0.137 mmol) was taken up in CH
2Cl
2 (2.5 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was stirred at
room temperature for 6 h. Toluene (3 mL) was added; the reaction mixture was concentrated
to dryness and then azeotroped with MeOH three times to provide the title compound
as a dark red powder (77 mg, 99%, TFA salt). Analytical HPLC and NMR indicated that
the material was >95% pure and did not require further purification prior to amide
coupling.
1H NMR (MeOD, 400 MHz) δ 8.40 (dd,
J = 8.2, 0.6 Hz, 1H), 8.37 (dd,
J = 8.2, 1.5 Hz, 1H), 7.94 (dd,
J = 1.5, 0.6 Hz, 1H), 7.06 (d,
J = 9.2 Hz, 2H), 6.61 (dd,
J= 9.2, 2.2 Hz, 2H), 6.55 (d,
J= 2.2 Hz, 2H), 4.31 (t,
J= 7.6 Hz, 8H), 2.56 (p,
J = 7.6 Hz, 4H);
19F NMR (MeOD, 376 MHz) δ -75.32 (s);
13C NMR (MeOD, 101 MHz) δ 167.7 (C), 167.5 (C), 160.1 (C), 158.7 (C), 158.0 (C), 136.2
(C), 135.9 (C), 135.4 (C), 132.8 (CH), 132.25 (CH), 132.24 (CH), 132.19 (CH), 114.8
(C), 113.6 (CH), 95.2 (CH), 52.9 (CH
2), 16.8 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
27H
23N
2O
5 [M+H]
+ 455.1601, found 455.1610.
Example 20. 2-(3,6-Di(azetidin-1-yl)xanthylium-9-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate
[0178]

[0179] 4-Carboxy-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate (Example 19; 20 mg, 35.2
µmol) was combined with DSC (19.8 mg, 77.4 µmol, 2.2 eq) in DMF (1.5 mL). After adding
Et
3N (14.7 µL, 106 µmol, 3 eq) and DMAP (0.4 mg, 3.52 µmol, 0.1 eq), the reaction was
stirred at room temperature for 2 h. Purification of the crude reaction mixture by
reverse phase HPLC (10-95% MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) afforded 18.3 mg (78%, TFA
salt) of the title compound as a dark purple solid.
1H NMR (DMSO-d
6, 400 MHz) δ 8.47 (dd,
J = 8.2, 1.8 Hz, 1H), 8.42 (d,
J = 8.2 Hz, 1H), 8.11 (d,
J = 1.6 Hz, 1H), 7.06 (d,
J = 9.1 Hz, 2H), 6.60 (d,
J = 9.1 Hz, 2H), 6.58 - 6.53 (m, 2H), 4.26 (t,
J= 7.4 Hz, 8H), 2.91 (s, 4H), 2.44 (p,
J= 7.7 Hz, 4H); Analytical HPLC: 97.4% purity (4.6 mm x 150 mm 5 µm C18 column; 5 µL
injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; UV detection at 550 nm); MS (ESI) calcd for C
31H
26N
3O
7 [M+H]
+ 552.2, found 552.0.
Example 21. 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate
[0180]

[0181] 4-Carboxy-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate (Example 19; 10 mg, 17.6
µmol) was combined with DSC (9.9 mg, 38.7 µmol, 2.2 eq) in DMF (1 mL). After adding
Et
3N (14.7 µL, 106 µmol, 6 eq) and DMAP (0.2 mg, 1.76 µmol, 0.1 eq), the reaction was
stirred at room temperature for 1 h while shielded from light. A solution of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine
("HaloTag(O2)amine," 9.8 mg, 44.0 µmol, 2.5 eq) in DMF (100 µL) was then added. The
reaction was stirred an additional 4 h at room temperature. It was subsequently diluted
with saturated NaHCO
3 and extracted with CH
2Cl
2 (2×). The combined organic extracts were dried (MgSO
4), filtered, deposited onto Celite, and concentrated
in vacuo. Silica gel chromatography (0-10% MeOH/CH
2Cl
2, linear gradient, with constant 1% v/v AcOH additive; dry load with Celite) followed
by reverse phase HPLC (10-95% MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) afforded 8.5 mg (62%, TFA
salt) of the title compound as a dark red solid.
1H NMR (MeOD, 400 MHz) δ 8.79 (t
, J = 5.4 Hz, 1H), 8.39
(d, J= 8.2 Hz, 1H), 8.20 (dd,
J = 8.2, 1.8 Hz, 1H), 7.80 (d,
J = 1.6 Hz, 1H), 7.07 (d,
J = 9.2 Hz, 2H), 6.61 (dd,
J = 9.2, 2.2 Hz, 2H), 6.56 (d,
J= 2.2 Hz, 2H), 4.31 (t,
J= 7.6 Hz, 8H), 3.68 - 3.55 (m, 8H), 3.53 (t,
J= 6.6 Hz, 2H), 3.43 (t,
J = 6.5 Hz, 2H), 2.56 (p,
J = 7.6 Hz, 4H), 1.77 - 1.66 (m, 2H), 1.56 - 1.27 (m, 6H);
19F NMR (MeOD, 376 MHz) δ -75.33 (s); Analytical HPLC: >99% purity (4.6 mm × 150 mm
5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C
37H
43ClN
3O
6 [M+H]
+ 660.2835, found 660.2844.
Example 22. 4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate
[0182]

[0183] 4-Carboxy-2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)benzoate (Example 19; 10 mg, 17.6
µmol) was combined with DSC (9.9 mg, 38.7 µmol, 2.2 eq) in DMF (1 mL). After adding
Et
3N (14.7 µL, 106 µmol, 6 eq) and DMAP (0.2 mg, 1.76 µmol, 0.1 eq), the reaction was
stirred at room temperature for 1 h while shielded from light. 6-((4-(Aminomethyl)benzyl)oxy)-9
H-purin-2-amine ("BG-NH
2," 11.9 mg, 44.0 µmol, 2.5 eq) was then added. The reaction was stirred an additional
2 h at room temperature. Purification of the crude reaction mixture by reverse phase
HPLC (10-95% MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) afforded 11.5 mg (80%, TFA
salt) of the title compound as a dark red solid.
1H NMR (MeOD, 400 MHz) δ 9.28 (t,
J = 5.8 Hz, 1H), 8.39 (d,
J = 8.3 Hz, 1H), 8.20 (dd,
J = 8.2, 1.8 Hz, 1H), 8.17 (s, 1H), 7.81 (d,
J = 1.7 Hz, 1H), 7.50 (d,
J = 8.1 Hz, 2H), 7.40 (d,
J = 8.2 Hz, 2H), 7.04 (d,
J= 9.2 Hz, 2H), 6.58 (dd,
J= 9.1, 2.2 Hz, 2H), 6.54 (d,
J= 2.1 Hz, 2H), 5.60 (s, 2H), 4.63 - 4.55 (m, 2H), 4.30 (t,
J = 7.6 Hz, 8H), 2.56 (p,
J = 7.7 Hz, 4H);
19F NMR (MeOD, 376 MHz) δ -75.44 (s); Analytical HPLC: 98.3% purity (4.6 mm × 150 mm
5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; UV detection at 550 nm); HRMS (ESI) calcd for C
40H
35N
8O
5 [M+H]
+ 707.2725, found 707.2723.
Example 23. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)xanthylium-9-yl)-4-(tert-butoxycarbonyl)benzoate
[0184]

[0185] The procedure described for Example 7 was used to prepare the title compound from
tert-butyl 3-oxo-3',6'-bis(((trifluoromethyl)sulfonyl)oxy)-3
H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate (Example 18, Step 2) and 3,3-difluoroazetidine
hydrochloride (84%, pink solid).
1H NMR (CDCl
3, 400 MHz) δ 8.21 (dd,
J = 8.0, 1.3 Hz, 1H), 8.04 (dd,
J = 8.0, 0.8 Hz, 1H), 7.73 (dd,
J = 1.2, 0.8 Hz, 1H), 6.61 (d,
J = 8.6 Hz, 2H), 6.30 (d,
J = 2.4 Hz, 2H), 6.17 (dd,
J = 8.6, 2.4 Hz, 2H), 4.25 (t,
3JHF = 11.7 Hz, 8H), 1.55 (s, 9H);
19F NMR (CDCl
3, 376 MHz) δ -100.06 (p,
3JFH = 11.7 Hz);
13C NMR (CDCl
3, 101 MHz) δ 168.8 (C), 164.3 (C), 153.3 (C), 152.6 (C), 151.4 (t,
4JCF = 2.9 Hz, C), 138.4 (C), 130.9 (CH), 130.2 (C), 129.3 (CH), 125.1 (CH), 125.0 (CH),
115.7 (t,
1JCF = 274.5 Hz, CF
2), 109.1 (C), 108.9 (CH), 99.4 (CH), 84.3 (C), 82.7 (C), 63.4 (t,
2JCF = 26.3 Hz, CH
2), 28.2 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
30-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); MS (ESI) calcd for C
31H
27F
4N
2O
5 [M+H]
+ 583.2, found 583.1.
Example 24. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)xanthylium-9-yl)-4-carboxybenzoate
[0186]

[0187] The procedure described for Example 19 was used to prepare the title compound from
Example 23 (93%, dark pink solid, TFA salt).
1H NMR (MeOD, 400 MHz) δ 8.44 (d,
J = 8.3 Hz, 1H), 8.40 (dd,
J= 8.2, 1.6 Hz, 1H), 7.99 - 7.96 (m, 1H), 7.23
(d, J= 9.1 Hz, 2H), 6.83
(d, J= 2.2 Hz, 2H), 6.79 (dd,
J = 9.1, 2.3 Hz, 2H), 4.70 (t,
3JHF = 11.6 Hz, 8H);
19F NMR (MeOD, 376 MHz) δ -75.59 (s, 3F), -100.90 (p,
3JFH = 11.6 Hz, 4F);
13C NMR (MeOD, 101 MHz) δ 167.6 (C), 167.3 (C), 159.1 (C), 157.7 (t,
4JCF = 3.9 Hz, C), 136.1 (C), 135.8 (C), 135.4 (C), 132.9 (CH), 132.7 (CH), 132.6 (CH),
132.1 (CH), 119.2 (C), 116.5 (t,
1JCF = 271.9 Hz, CF
2), 116.1 (C), 115.2 (CH), 97.4 (CH), 64.2 (t,
2JCF = 29.1 Hz, CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); MS (ESI) calcd for C
27H
19F
4N
2O
5 [M+H]
+ 527.1, found 527.0.
Example 25. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)xanthylium-9-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate
[0188]

[0189] The procedure described for Example 21 was used to prepare the title compound from
Example 24 (62%, pink solid).
1H NMR (CDCl
3, 400 MHz) δ 8.05 (dd,
J = 8.0, 0.8 Hz, 1H), 7.98 (dd,
J = 8.0, 1.4 Hz, 1H), 7.55 (dd,
J = 1.5, 0.8 Hz, 1H), 6.72 (s, 1H), 6.62 (d,
J = 8.6 Hz, 2H), 6.31 (d,
J = 2.4 Hz, 2H), 6.17 (dd,
J = 8.6, 2.4 Hz, 2H), 4.26 (t,
3JHF = 11.7 Hz, 8H), 3.69 - 3.57 (m, 6H), 3.56 - 3.48 (m, 4H), 3.40 (t,
J= 6.6 Hz, 2H), 1.79 - 1.70 (m, 2H), 1.54 - 1.48 (m, 2H), 1.46 - 1.38 (m, 2H), 1.37
- 1.29 (m, 2H);
19F NMR (CDCl
3, 376 MHz) δ -100.04 (p,
3JFH = 11.8 Hz, 4F); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5
µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 550 nm); MS (ESI) calcd for C
37H
39ClF
4N
3O
6 [M+H]
+ 732.2, found 732.1.
Example 26. 2-(3,6-Di(azetidin-1-yl)xanthylium-9-yl)-4-(methoxycarbonyl)benzoate
[0190]

[0191] Step 1: 3',6'-Dibromo-3-oxo-3
H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylic acid (1.50 g, 2.99 mmol;
Woodroofe, C. C.; Lim, M. H.; Bu, W.; Lippard, S. J. Tetrahedron 2005, 61, 3097) was suspended in MeOH (50 mL), and H
2SO
4 (293 mg, 2.99 mmol, 1 eq) was added. The reaction was stirred at reflux for 72 h.
It was subsequently concentrated
in vacuo, and the resulting residue was diluted with saturated NaHCO
3 and extracted with 15%
i-PrOH/CHCl
3 (2×). The combined organic extracts were dried (MgSO
4), filtered, and evaporated. Silica gel chromatography (0-10% EtOAc/hexanes, linear
gradient, with constant 40% v/v CH
2Cl
2) yielded 1.49 g (97%) of methyl 3',6'-dibromo-3-oxo-3
H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxylate as a white solid.
1H NMR (CDCl
3, 400 MHz) δ 8.31 (dd,
J= 8.0, 1.3 Hz, 1H), 8.10
(dd, J= 8.0, 0.7 Hz, 1H), 7.76 (dd,
J = 1.2, 0.8 Hz, 1H), 7.52 (d,
J = 1.9 Hz, 2H), 7.20 (dd,
J = 8.5, 1.9 Hz, 2H), 6.68 (d,
J = 8.5 Hz, 2H), 3.89 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 168.1 (C), 165.3 (C), 153.1 (C), 151.2 (C), 137.0 (C), 131.6 (CH), 129.24
(C), 129.21 (CH), 127.8 (CH), 125.7 (CH), 125.1 (CH), 124.6 (C), 120.7 (CH), 117.4
(C), 81.5 (C), 53.0 (CH
3); MS (ESI) calcd for C
22H
13Br
2O
5 [M+H]
+ 514.9, found 515.1.
[0192] Step 2: The procedure described for Example 7 was used to prepare the title compound 2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)-4-(methoxycarbonyl)benzoate
from the bromide synthesized in Step 1 (86%, red solid).
1H NMR (MeOD, 400 MHz) 8 8.24 (dd,
J= 8.1, 1.7 Hz, 1H), 8.12 (dd,
J= 8.1, 0.4 Hz, 1H), 7.83 - 7.80 (m, 1H), 7.16 (d,
J = 9.2 Hz, 2H), 6.56 (dd,
J = 9.2, 2.2 Hz, 2H), 6.48 (d,
J = 2.2 Hz, 2H), 4.32 - 4.22 (m, 8H), 3.90 (s, 3H), 2.54 (p,
J = 7.6 Hz, 4H); MS (ESI) calcd for C
28H
25N
2O
5 [M+H]
+ 469.2, found 469.2.
Example 27. Methyl 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate
[0193]

[0194] To a solution of 2-(3,6-di(azetidin-1-yl)xanthylium-9-yl)-4-(methoxycarbonyl)benzoate
(Example 26; 135 mg, 0.288 mmol) in CH
2Cl
2 (9 mL) was added oxalyl chloride (98 µL, 1.15 mmol, 4 eq). After stirring the reaction
at room temperature for 2 h, it was concentrated to dryness. The residue was redissolved
in CH
2Cl
2 (9 mL); Et
3N (50 µL, 0.360 mmol, 1.25 eq) and (trimethylsilyl)diazomethane (2.0 M in Et
2O, 252 µL, 0.504 mmol, 1.75 eq) were then added in succession. The reaction was stirred
at room temperature for 90 min, concentrated
in vacuo, and purified twice by flash chromatography on silica gel (0-50% EtOAc/hexanes, linear
gradient; then, 0-25% EtOAc/toluene, linear gradient) to afford 40 mg (28%) of the
title compound as a yellow solid.
1H NMR (CDCl
3, 400 MHz) δ 8.07 (dd,
J= 8.0, 1.4 Hz, 1H), 7.87 (dd,
J = 8.0, 0.6 Hz, 1H), 7.68 (dd,
J= 1.4, 0.6 Hz, 1H), 6.66 (d,
J= 8.5 Hz, 2H), 6.18 (d,
J= 2.3 Hz, 2H), 6.07 (dd,
J= 8.5, 2.4 Hz, 2H), 3.96 - 3.84 (m, 8H), 3.82 (s, 3H), 2.37 (p,
J= 7.2 Hz, 4H); MS (ESI) calcd for C
29H
25N
4O
4 [M+H]
+ 493.2, found 493.3.
Example 28. 2,5-Dioxopyrrolidin-1-yl 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate
[0195]

[0196] Step 1: To a solution of methyl 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate
(Example 27; 40 mg, 81.2 µmol) in 2:1 MeOH/THF (6 mL) under nitrogen was added 1 M
NaOH (203 µL, 0.203 mmol, 2.5 eq). After stirring the solution at room temperature
for 2 h, additional 1 M NaOH (203 µL, 0.203 mmol, 2.5 eq) was added. The reaction
was stirred at room temperature for 24 h. It was subsequently acidified with 1 M HCl
(420 µL), diluted with water, and extracted with CH
2Cl
2 (2×). The organic extracts were dried (MgSO
4), filtered, and concentrated
in vacuo to provide 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylic
acid (37 mg, 95%) as a yellow solid. MS (ESI) calcd for C
28H
23N
4O
4 [M]
+ 479.2, found 479.3.
[0197] Step 2: The acid from Step 1 (37 mg, 77.3 µmol) was combined with TSTU (35 mg, 0.116 mmol,
1.5 eq) in DMF (2 mL), and DIEA (40 µL, 0.232 mmol, 3 eq) was added. After stirring
the reaction at room temperature for 1 h, it was concentrated to dryness and deposited
onto Celite. Flash chromatography on silica gel (10-100% EtOAc/hexanes, linear gradient;
dry load with Celite) afforded the title compound 2,5-dioxopyrrolidin-1-yl 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate
as a yellow-orange solid (30 mg, 68%).
1H NMR (CDCl
3, 400 MHz) δ 8.16 (dd,
J = 8.0, 1.5 Hz, 1H), 7.93 (dd,
J= 8.0, 0.6 Hz, 1H), 7.75 (dd,
J= 1.4, 0.6 Hz, 1H), 6.66 (d,
J = 8.5 Hz, 2H), 6.16 (d,
J= 2.3 Hz, 2H), 6.09 (dd,
J= 8.5, 2.4 Hz, 2H), 3.90 (t,
J= 7.3 Hz, 8H), 2.86 (s, 4H), 2.37 (p,
J = 7.2 Hz, 4H); MS (ESI) calcd for C
32H
26N
5O
6 [M+H]
+ 576.2, found 576.3.
Example 29. 3',6'-Di(azetidin-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxamide
[0198]

[0199] 2,5-Dioxopyrrolidin-1-yl 3',6'-di(azetidin-1-yl)-2-diazo-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-6-carboxylate
(Example 28; 15 mg, 26.1 µmol) was dissolved in DMF (1 mL). A solution of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine
("HaloTag(O2)amine," 11.7 mg, 52.2 µmol, 2 eq) in DMF (250 µL) was added, followed
by DIEA (22.7 µL, 0.131 mmol, 5 eq). After stirring the reaction at room temperature
for 2 h, it was concentrated to dryness and purified by silica gel chromatography
(0-100% EtOAc/toluene, linear gradient) to provide the title compound as a yellow
foam (15.9 mg, 89%).
1H NMR (CDCl
3, 400 MHz) δ 7.86 (dd,
J = 7.9, 0.6 Hz, 1H), 7.79 (dd,
J = 8.0, 1.5 Hz, 1H), 7.43 (dd,
J = 1.4, 0.6 Hz, 1H), 6.66 (d,
J = 8.5 Hz, 2H), 6.59 (t,
J = 5.1 Hz, 1H), 6.16 (d,
J = 2.3 Hz, 2H), 6.07
(dd, J= 8.5, 2.4 Hz, 2H), 3.95 - 3.83 (m, 8H), 3.64 - 3.48 (m, 10H), 3.39 (t,
J = 6.6 Hz, 2H), 2.37 (p,
J = 7.2 Hz, 4H), 1.78 - 1.69 (m, 2H), 1.55 - 1.48 (m, 2H), 1.46 - 1.36 (m, 2H), 1.36
- 1.27 (m, 2H); MS (ESI) calcd for C
38H
43ClN
5O
5 [M+H]
+ 684.3, found 684.4.
Example 30. 2-(3,7-Di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate
[0200]

[0201] Step 1: A vial was charged with tert-butyl 2-bromobenzoate (309 mg, 1.20 mmol, 1.5 eq), sealed,
and flushed with nitrogen. After dissolving the bromide in THF (2 mL) and cooling
the reaction to -15 °C,
iPrMgCl·LiCl (1.3 M in THF, 924 µL, 1.20 mmol, 1.5 eq) was added. The reaction was
warmed to -5 °C and stirred for 5 h. A solution of 3,7-bis((
tert-butyldimethylsilyl)oxy)-5,5-dimethyldibenzo[
b,e]silin-10(5
H)-one (400 mg, 0.802 mmol; from Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai,
T.; Nagano, T. Chem. Commun., 2011, 47, 4162) in THF (2 mL) was then added dropwise.
After stirring for 10 min at -5 °C, the reaction mixture was warmed to room temperature
and stirred for 30 min. It was subsequently quenched with saturated NH
4Cl, diluted with water, and extracted with EtOAc (2×). The combined organics were
washed with brine, dried (MgSO
4), filtered, and evaporated. Silica gel chromatography (0-20% Et
2O/hexanes, linear gradient) provided 271 mg (56%) of 3,7-bis((
tert-butyldimethylsilyl)oxy)-5,5-dimethyl-3'
H,5
H-spiro[dibenzo[
b,e]siline-10,1'-isobenzofuran]-3'-one as a colorless gum.
1H NMR (CDCl
3, 400 MHz) δ 7.97 (dt,
J= 7.5, 0.9 Hz, 1H), 7.66 (td,
J= 7.5, 1.2 Hz, 1H), 7.56 (td,
J= 7.5, 0.9 Hz, 1H), 7.35 - 7.29 (m, 1H), 7.12 (d,
J= 2.7 Hz, 2H), 6.85 (d,
J= 8.7 Hz, 2H), 6.67 (dd,
J = 8.7, 2.7 Hz, 2H), 0.97 (s, 18H), 0.62 (s, 3H), 0.60 (s, 3H), 0.19 (s, 12H);
13C NMR (CDCl
3, 101 MHz) δ 170.5 (C), 155.3 (C), 154.0 (C), 137.8 (C), 137.2 (C), 134.0 (CH), 129.1
(CH), 128.6 (CH), 126.6 (C), 126.1 (CH), 125.1 (CH), 124.7 (CH), 121.2 (CH), 90.8
(C), 25.8 (CH
3), 18.4 (C), 0.2 (CH
3), -1.5 (CH
3), -4.21 (CH
3), -4.23 (CH
3); HRMS (ESI) calcd for C
34H
47O
4Si
3 [M+H]
+ 603.2777, found 603.2771.
[0202] Step 2: To a solution of the product from Step 1 (194 mg, 0.322 mmol) in THF (5 mL) at 0
°C was added TBAF (1.0 M in THF, 965 µL, 0.965 mmol, 3 eq). The reaction was stirred
at 0 °C for 10 min. It was subsequently diluted at 0 °C with saturated NH
4Cl and extracted with EtOAc (2×). The organic extracts were dried (MgSO
4), filtered, evaporated, and deposited onto silica gel. Flash chromatography (20-100%
EtOAc/hexanes, linear gradient, with constant 1% v/v AcOH additive; dry load with
silica gel) yielded 3,7-dihydroxy-5,5-dimethyl-3'
H,5
H-spiro[dibenzo[
b,e]siline-10,1'-isobenzofuran]-3'-one (120 mg, 99%) as an off-white solid.
1H NMR (MeOD, 400 MHz) δ 7.95 (d,
J= 7.7 Hz, 1H), 7.77 (td,
J= 7.6, 1.1 Hz, 1H), 7.65 (td,
J = 7.6, 0.7 Hz, 1H), 7.32 (d,
J = 7.7 Hz, 1H), 7.13 (d,
J = 2.7 Hz, 2H), 6.74 (d,
J = 8.7 Hz, 2H), 6.65 (dd,
J = 8.7, 2.7 Hz, 2H), 0.61 (s, 3H), 0.55 (s, 3H);
13C NMR (MeOD, 101 MHz) δ 172.6 (C), 158.3 (C), 155.8 (C), 138.8 (C), 136.3 (C), 135.6
(CH), 130.4 (CH), 129.6 (CH), 127.4 (C), 126.6 (CH), 125.8 (CH), 121.1 (CH), 117.7
(CH), 92.9 (C), 0.2 (CH
3), -1.6 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; UV detection at 254 nm); HRMS (ESI) calcd for C
22H
9O
4Si [M+H]
+ 375.1047, found 375.1047.
[0203] Step 3: The intermediate from Step 2 (120 mg, 0.320 mmol) was taken up in CH
2Cl
2 (5 mL) and cooled to 0 °C. Pyridine (207 µL, 2.56 mmol, 8.0 eq) and trifluoromethanesulfonic
anhydride (216 µL, 1.28 mmol, 4.0 eq) were added, and the ice bath was removed. The
reaction was stirred at room temperature for 2 h. It was subsequently diluted with
water and extracted with CH
2Cl
2 (2×). The combined organic extracts were washed with brine, dried (MgSO
4), filtered, and concentrated
in vacuo. Flash chromatography on silica gel (0-30% EtOAc/hexanes, linear gradient) afforded
172 mg (84%) of 5,5-dimethyl-3'-oxo-3'
H,5
H-spiro[dibenzo[
b,e]siline-10,1'-isobenzofuran]-3,7-diyl bis(trifluoromethanesulfonate) as a colorless
foam.
1H NMR (CDCl
3, 400 MHz) δ 8.04 (dt,
J = 7.7, 0.9 Hz, 1H), 7.77 (td,
J= 7.5, 1.2 Hz, 1H), 7.66 (td,
J= 7.5, 0.8 Hz, 1H), 7.57 (dd,
J= 2.4, 0.5 Hz, 2H), 7.38 (dt,
J = 7.6, 0.7 Hz, 1H), 7.185 (AB of ABX, □A = 2878.9,
JAx = 0.3, □B = 2871.0,
JBx = 2.8,
JAB = 8.9 Hz, 4H), 0.75 (s, 3H), 0.72 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -73.30;
13C NMR (CDCl
3, 101 MHz) δ 169.2 (C), 151.8 (C), 149.5 (C), 144.3 (C), 139.3 (C), 134.8 (CH), 130.3
(CH), 129.2 (CH), 127.0 (CH), 126.5 (CH), 126.0 (C), 124.6 (CH), 122.8 (CH), 118.9
(CF
3,
1JCF = 320.8 Hz), 88.7 (C), 0.1 (CH
3), -1.7 (CH
3); HRMS (ESI) calcd for C
24H
17F
6O
8S
2Si [M+H]
+ 639.0033, found 639.0030.
[0204] Step 4: The procedure described for Example 7 was used to prepare the title compound 2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[
b,e]silin-10-ylium-10(5
H)-yl)benzoate from the ditriflate synthesized in Step 3 (92%, off-white solid).
1H NMR (CDCl
3, 400 MHz) δ 7.98 - 7.93 (m, 1H), 7.63 (td,
J= 7.5, 1.2 Hz, 1H), 7.53 (td,
J= 7.5, 0.9 Hz, 1H), 7.32 - 7.28 (m, 1H), 6.75
(d, J= 8.7 Hz, 2H), 6.66 (d,
J = 2.6 Hz, 2H), 6.25 (dd,
J= 8.7, 2.7 Hz, 2H), 3.89 (t,
J= 7.2 Hz, 8H), 2.36 (p,
J= 7.2 Hz, 4H), 0.60 (s, 3H), 0.58 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 170.7 (C), 154.3 (C), 151.0 (C), 137.1 (C), 133.7 (CH), 132.9 (C), 128.8
(CH), 128.0 (CH), 127.2 (C), 125.8 (CH), 124.8 (CH), 115.7 (CH), 112.3 (CH), 92.1
(C), 52.4 (CH
2), 17.1 (CH
2), 0.5 (CH
3), -1.5 (CH
3); Analytical HPLC: 98.7% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C
28H
29N
2O
2Si [M+H]
+ 453.1993, found 453.1998.
Example 31. 2-(3,7-Bis(3-fluoroazetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate
[0205]

[0206] The procedure described for Example 7 was used to prepare the title compound from
5,5-dimethyl-3'-oxo-3'
H,5
H-spiro[dibenzo[
b,
e]siline-10,1'-isobenzofuran]-3,7-diyl bis(trifluoromethanesulfonate) (Example 30,
Step 3) and 3-fluoroazetidine hydrochloride (78%, off-white solid).
1H NMR (CDCl
3, 400 MHz) δ 7.97 (dt,
J = 7.6, 0.9 Hz, 1H), 7.65 (td,
J = 7.5, 1.2 Hz, 1H), 7.55 (td,
J = 7.5, 0.9 Hz, 1H), 7.29 (dt,
J = 7.7, 0.8 Hz, 1H), 6.80 (d,
J = 8.7 Hz, 2H), 6.70 (d,
J = 2.6 Hz, 2H), 6.30 (dd,
J = 8.7, 2.7 Hz, 2H), 5.41 (dtt,
2JHF = 57.0 Hz,
J = 5.9, 3.7 Hz, 2H), 4.25 - 4.14 (m, 4H), 4.04 - 3.91 (m, 4H), 0.62 (s, 3H), 0.60
(s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -180.48 (dtt,
JFH = 57.0, 23.9, 18.2 Hz);
13C NMR (CDCl
3, 101 MHz) δ 170.6 (C), 154.1 (C), 150.0 (d,
4JCF = 1.0 Hz, C), 137.2 (C), 133.93 (C), 133.86 (CH), 129.0 (CH), 128.1 (CH), 127.0 (C),
126.0 (CH), 124.7 (CH), 116.3 (CH), 112.9 (CH), 91.6 (C), 82.8 (d,
1JCF = 204.8 Hz, CFH), 59.6 (d,
2JCF = 23.8 Hz, CH
2), 0.5 (CH
3), -1.4 (CH
3); Analytical HPLC: 98.7% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
30-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 650 nm); MS (ESI) calcd for C
28H
27F
2N
2O
2Si [M+H]
+ 489.2, found 489.1.
Example 32. 4-(tert-Butoxycarbonyl)-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate
[0207]

[0208] Step 1: A suspension of 2-bromoterephthalic acid (2.50 g, 10.2 mmol) in toluene (25 mL) was
heated to 80 °C, and
N,N-dimethylformamide di-
tert-butyl acetal (24.5 mL, 102 mmol, 10 eq) was added dropwise over 15 min. The reaction
was stirred at 80 °C for 30 min. After cooling the mixture to room temperature, it
was diluted with saturated NaHCO
3 and extracted with EtOAc (2×). The combined organic extracts were washed with water
and brine, dried (MgSO
4), filtered, and evaporated. Flash chromatography (0-10% Et
2O/hexanes, linear gradient) provided di-
tert-butyl 2-bromoterephthalate as a colorless gum (3.29 g, 90%).
1H NMR (CDCl
3, 400 MHz) δ 8.19 (d,
J = 1.4 Hz, 1H), 7.92 (dd,
J = 8.0, 1.6 Hz, 1H), 7.67 (d,
J = 8.0 Hz, 1H), 1.62 (s, 9H), 1.60 (s, 9H);
13C NMR (CDCl
3, 101 MHz) δ 165.4 (C), 163.8 (C), 138.0 (C), 135.1 (C), 134.9 (CH), 130.4 (CH), 128.1
(CH), 120.7 (C), 83.3 (C), 82.3 (C), 28.26 (CH
3), 28.25 (CH
3); HRMS (ESI) calcd for C
6H
21BrO
4Na [M+Na]
+ 379.0515, found 379.0531.
[0209] Step 2: A vial was charged with the product of Step 1 (537 mg, 1.50 mmol, 1.5 eq), sealed,
and flushed with nitrogen. After dissolving the bromide in THF (2.5 mL) and cooling
the reaction to -50 °C,
iPrMgCl·LiCl (1.3 M in THF, 1.16 mL, 1.50 mmol, 1.5 eq) was added. The reaction was
warmed to -40 °C and stirred for 2 h. A solution of 3,7-bis((
tert-butyldimethylsilyl)oxy)-5,5-dimethyldibenzo[
b,
e]silin-10(5
H)-one (500 mg, 1.00 mmol; from
Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. Chem. Commun.,
2011, 47, 4162) in THF (2.5 mL) was then added dropwise. The reaction mixture was warmed to room
temperature and stirred for 2 h. It was subsequently quenched with saturated NH
4Cl, diluted with water, and extracted with EtOAc (2×). The combined organics were
washed with brine, dried (MgSO
4), filtered, and evaporated. Silica gel chromatography (0-10% Et
2O/hexanes, linear gradient) provided 213 mg (30%) of
tert-butyl 3,7-bis((tertbutyldimethylsilyl)oxy)-5,5-dimethyl-3'-oxo-3'
H,5
H-spiro[dibenzo[
b,
e]siline-10,1'-isobenzofuran]-6'-carboxylate as a colorless solid.
1H NMR (CDCl
3, 400 MHz) δ 8.13 (dd,
J = 8.0, 1.3 Hz, 1H), 7.98 (dd,
J = 8.0, 0.7 Hz, 1H), 7.84 (dd,
J = 1.2, 0.8 Hz, 1H), 7.13 (d,
J = 2.7 Hz, 2H), 6.93 (d,
J = 8.7 Hz, 2H), 6.72 (dd,
J = 8.7, 2.7 Hz, 2H), 1.56 (s, 9H), 0.98 (s, 18H), 0.67 (s, 3H), 0.59 (s, 3H), 0.196
(s, 6H), 0.194 (s, 6H);
13C NMR (CDCl
3, 101 MHz) δ 170.1 (C), 164.3 (C), 155.4 (C), 155.0 (C), 137.5 (C), 136.9 (C), 136.8
(C), 130.2 (CH), 128.7 (C), 128.3 (CH), 125.9 (CH), 125.2 (CH), 125.1 (CH), 121.6
(CH), 90.6 (C), 82.5 (C), 28.2 (CH
3), 25.8 (CH
3), 18.4 (C), -0.1 (CH
3), -0.7 (CH
3), -4.21 (CH
3), -4.23 (CH
3); HRMS (ESI) calcd for C
39H
55O
6Si
3 [M+H]
+ 703.3301, found 703.3311.
[0210] Step 3: To a solution of the product from Step 2 (205 mg, 0.292 mmol) in THF (5 mL) at 0
°C was added TBAF (1.0 M in THF, 1.17 □ L, 1.17 mmol, 4 eq). The reaction was stirred
at 0 °C for 10 min. It was subsequently diluted with saturated NH
4Cl and extracted with EtOAc (2×). The organic extracts were washed with brine, dried
(MgSO
4), filtered, and evaporated to provide an orange residue. The crude intermediate was
taken up in CH
2Cl
2 (5 mL) and cooled to 0 °C. Pyridine (189 µL, 2.33 mmol, 8 eq) and trifluoromethanesulfonic
anhydride (196 µL, 1.17 mmol, 4 eq) were added, and the ice bath was removed. The
reaction was stirred at room temperature for 2 h. It was then diluted with water and
extracted with CH
2Cl
2 (2×). The combined organics were washed with brine, dried (MgSO
4), filtered, and concentrated
in vacuo. Flash chromatography on silica gel (0-20% EtOAc/hexanes, linear gradient) afforded
209 mg (97%) of
tert-butyl 5,5-dimethyl-3'-oxo-3,7-bis(((trifluoromethyl)sulfonyl)oxy)-3'
H,5
H-spiro[dibenzo[
b,
e]siline-10,1'-isobenzofuran]-6'-carboxylate as a colorless solid.
1H NMR (CDCl
3, 400 MHz) δ 8.21 (dd,
J = 8.0, 1.3 Hz, 1H), 8.05 (dd,
J = 8.0, 0.7 Hz, 1H), 7.93 - 7.90 (m, 1H), 7.58 (d,
J = 2.6 Hz, 2H), 7.28 (d,
J = 8.9 Hz, 2H), 7.22 (dd,
J = 8.9, 2.7 Hz, 2H), 1.58 (s, 9H), 0.81 (s, 3H), 0.71 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -73.28 (s);
13C NMR (CDCl
3, 101 MHz) δ 168.9 (C), 163.8 (C), 152.8 (C), 149.5 (C), 144.1 (C), 138.3 (C), 138.2
(C), 131.2 (CH), 128.8 (CH), 128.0 (C), 126.8 (CH), 126.6 (CH), 124.8 (CH), 123.2
(CH), 118.9 (q,
1JCF = 320.8 Hz, CF
3), 88.6 (C), 83.1 (C), 28.2 (CH
3), -0.1 (CH
3), -0.9 (CH
3); HRMS (ESI) calcd for C
29H
25F
6O
10S
2Si [M+H]
+ 739.0557, found 739.0555.
[0211] Step 4: The procedure described for Example 7 was used to prepare the title compound 4-(
tert-butoxycarbonyl)-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[
b,e]silin-10-ylium-10(5
H)-yl)benzoate from the ditriflate synthesized in Step 3 (91%, off-white foam).
1H NMR (CDCl
3, 400 MHz) δ 8.11 (dd,
J = 8.0, 1.3 Hz, 1H), 7.95 (dd,
J = 8.0, 0.7 Hz, 1H), 7.82 (dd,
J = 1.2, 0.8 Hz, 1H), 6.82 (d,
J = 8.7 Hz, 2H), 6.66 (d,
J= 2.6 Hz, 2H), 6.29 (dd,
J= 8.7, 2.7 Hz, 2H), 3.90 (t,
J= 7.3 Hz, 8H), 2.36 (p,
J= 7.2 Hz, 4H), 1.54 (s, 9H), 0.64 (s, 3H), 0.58 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 170.3 (C), 164.5 (C), 155.4 (C), 151.0 (C), 137.2 (C), 136.2 (C), 132.4
(C), 129.9 (CH), 129.2 (C), 127.7 (CH), 125.6 (CH), 125.2 (CH), 115.6 (CH), 112.6
(CH), 91.9 (C), 82.3 (C), 52.3 (CH
2), 28.2 (CH
3), 17.0 (CH
2), 0.2 (CH
3), -0.7 (CH
3); HRMS (ESI) calcd for C
33H
37N
2O
4Si [M+H]
+ 553.2517, found 553.2529.
Example 33. 4-Carboxy-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate
[0212]

[0213] The procedure described for Example 19 was used to prepare the title compound from
Example 32 (99%, dark blue-green solid, TFA salt).
1H NMR (MeOD, 400 MHz) δ 8.30 - 8.23 (m, 2H), 7.82 - 7.78 (m, 1H), 6.90 (d,
J = 2.5 Hz, 2H), 6.86 (d,
J= 9.2 Hz, 2H), 6.33 (dd,
J= 9.2, 2.5 Hz, 2H), 4.27 (t,
J= 7.4 Hz, 8H), 2.51 (p,
J= 7.6 Hz, 4H), 0.60 (s, 3H), 0.53 (s, 3H);
19F NMR (MeOD, 376 MHz) δ - 75.45 (s); Analytical HPLC: 98.7% purity (4.6 mm × 150 mm
5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C
29H
29N
2O
4Si [M+H]
+ 497.1891, found 497.1890.
Example 34. 2-(3,7-Di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate
[0214]

[0215] 4-Carboxy-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[
b,
e]silin-10-ylium-10(5
H)-yl)benzoate (Example 33; 40 mg, 65.5 µmol) was combined with DSC (37 mg, 144 µmol,
2.2 eq) in DMF (2.5 mL). After adding Et
3N (55 µL, 393 µmol, 6 eq) and DMAP (0.8 mg, 6.55 µmol, 0.1 eq), the reaction was stirred
at room temperature for 3 h. It was subsequently diluted with 10% w/v citric acid
and extracted with EtOAc (2×). The combined organic extracts were washed with brine,
dried (MgSO
4), filtered, and concentrated
in vacuo. Flash chromatography (0-50% EtOAc/toluene, linear gradient) yielded 31 mg (80%) of
the title compound as a yellow-green solid.
1H NMR (CDCl
3, 400 MHz) δ 8.27 (dd,
J = 8.0, 1.4 Hz, 1H), 8.07 (dd,
J = 8.0, 0.7 Hz, 1H), 8.00 (dd,
J = 1.3, 0.7 Hz, 1H), 6.74 (d,
J = 8.7 Hz, 2H), 6.66 (d,
J = 2.6 Hz, 2H), 6.30 (dd,
J = 8.7, 2.7 Hz, 2H), 3.91 (t,
J = 7.3 Hz, 8H), 2.89 (s, 4H), 2.37 (p,
J = 7.2 Hz, 4H), 0.62 (s, 3H), 0.56 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 169.4 (C), 169.0 (C), 161.1 (C), 155.5 (C), 151.2 (C), 136.5 (C), 131.7
(C), 131.6 (C), 130.7 (CH), 130.1 (C), 127.8 (CH), 126.8 (CH), 126.3 (CH), 115.8 (CH),
112.7 (CH), 92.4 (C), 52.3 (CH
2), 25.8 (CH
2), 17.0 (CH
2), 0.3 (CH
3), -1.1 (CH
3); HRMS (ESI) calcd for C
33H
32N
3O
6Si [M+H]
+ 594.2055, found 594.2069.
Example 35. 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)benzoate
[0216]

[0217] 4-Carboxy-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[
b,
e]silin-10-ylium-10(5
H)-yl)benzoate (Example 33; 30 mg, 49.1 µmol) was combined with DSC (28 mg, 108 µmol,
2.2 eq) in DMF (2 mL). After adding Et
3N (41 µL, 295 µmol, 6 eq) and DMAP (0.6 mg, 4.91 µmol, 0.1 eq), the reaction was stirred
at room temperature for 1 h while shielded from light. A solution of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine
("HaloTag(02)amine," 27 mg, 123 µmol, 2.5 eq) in DMF (250 µL) was then added. The
reaction was stirred an additional 2 h at room temperature. It was subsequently diluted
with saturated NaHCO
3 and extracted with EtOAc (2×). The combined organic extracts were washed with water
and brine, dried (MgSO
4), filtered, and concentrated
in vacuo. Silica gel chromatography (10-100% EtOAc/toluene, linear gradient) afforded 25 mg
(73%) of the title compound as a blue foam.
1H NMR (CDCl
3, 400 MHz) δ 7.98 (dd,
J = 8.0, 0.7 Hz, 1H), 7.90 (dd,
J = 8.0, 1.4 Hz, 1H), 7.68 (dd,
J = 1.2, 0.7 Hz, 1H), 6.75 (d,
J = 8.7 Hz, 2H), 6.74 - 6.68 (m, 1H), 6.66 (d,
J = 2.6 Hz, 2H), 6.26 (dd,
J = 8.7, 2.7 Hz, 2H), 3.89 (t,
J = 7.3 Hz, 8H), 3.67 - 3.60 (m, 6H), 3.56 - 3.53 (m, 2H), 3.50 (t,
J = 6.7 Hz, 2H), 3.39 (t,
J = 6.7 Hz, 2H), 2.36 (p,
J = 7.2 Hz, 4H), 1.78 - 1.68 (m, 2H), 1.56 - 1.47 (m, 2H), 1.44 - 1.35 (m, 2H), 1.35
- 1.25 (m, 2H), 0.63 (s, 3H), 0.57 (s, 3H); Analytical HPLC: >99% purity (4.6 mm ×
150 mm 5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; UV detection at 650 nm); HRMS (ESI) calcd for C
39H
49ClN
3O
5Si [M+H]
+ 702.3125, found 702.3137.
Example 36. 4-((4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)carbamoyl)-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[b,e]silm-10-ylium-10(5H)-yl)benzoate
[0218]

[0219] 4-Carboxy-2-(3,7-di(azetidin-1-yl)-5,5-dimethyldibenzo[
b,
e]silin-10-ylium-10(5
H)-yl)benzoate (Example 33; 25 mg, 40.9 µmol) was combined with DSC (23.1 mg, 90.1
µmol, 2.2 eq) in DMF (2 mL). After adding Et
3N (34 µL, 246 µmol, 6 eq) and DMAP (0.5 mg, 4.09 µmol, 0.1 eq), the reaction was stirred
at room temperature for 1 h while shielded from light. 6-((4-(Aminomethyl)benzyl)oxy)-9
H-purin-2-amine ("BG-NH
2," 28 mg, 102 µmol, 2.5 eq) was then added. The reaction was stirred an additional
2 h at room temperature. It was subsequently diluted with saturated NaHCO
3 and extracted with CH
2Cl
2 (2×). The combined organic extracts were dried (MgSO
4), filtered, and concentrated
in vacuo. Silica gel chromatography (0-10% MeOH/EtOAc, linear gradient) afforded 24.7 mg (80%)
of the title compound as a blue solid.
1H NMR (MeOD, 400 MHz) δ 8.02 (dd,
J = 8.0, 1.3 Hz, 1H), 7.99 (dd,
J = 8.0, 0.7 Hz, 1H), 7.82 (s, 1H), 7.67 - 7.64 (m, 1H), 7.46 (d,
J = 8.1 Hz, 2H), 7.32 (d,
J = 8.2 Hz, 2H), 6.73 (d,
J = 2.6 Hz, 2H), 6.70 (d,
J = 8.7 Hz, 2H), 6.32 (dd,
J = 8.7, 2.6 Hz, 2H), 5.51 (s, 2H), 4.52 (s, 2H), 3.87 (t,
J = 7.3 Hz, 8H), 2.35 (p,
J = 7.1 Hz, 4H), 0.58 (s, 3H), 0.51 (s, 3H); Analytical HPLC: >99% purity (4.6 mm x
150 mm 5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; UV detection at 650 nm); HRMS (ESI) calcd for C
42H
41N
8O
4Si [M+H]
+ 749.3015, found 749.2971.
Example 37. 2-(3,7-Bis(3-fluoroazetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-1-4-tert-butoxcarbonlbenzoate
[0220]

[0221] The procedure described for Example 7 was used to prepare the title compound from
tert-butyl 5,5-dimethyl-3'-oxo-3,7-bis(((trifluoromethyl)sulfonyl)oxy)-3'
H,5
H-spiro[dibenzo[
b,
e]siline-10,1'-isobenzofuran]-6'-carboxylate (Example 32, Step 3) and 3-fluoroazetidine
hydrochloride (85%, off-white solid).
1H NMR (CDCl
3, 400 MHz) δ 8.12 (dd,
J = 8.0, 1.3 Hz, 1H), 7.97 (dd,
J = 7.9, 0.8 Hz, 1H), 7.82 (dd,
J = 1.3, 0.8 Hz, 1H), 6.88 (d,
J = 8.7 Hz, 2H), 6.70 (d,
J = 2.6 Hz, 2H), 6.35 (dd,
J = 8.7, 2.7 Hz, 2H), 5.41 (dtt,
2JHF = 57.0, 5.9, 3.7 Hz, 2H), 4.26 - 4.15 (m, 4H), 4.05 - 3.93 (m, 4H), 1.55 (s, 9H),
0.67 (s, 3H), 0.60 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -180.48 (dtt,
JFH = 57.0, 23.9, 18.2 Hz);
13C NMR (CDCl
3, 101 MHz) δ 170.2 (C), 164.4 (C), 155.1 (C), 150.0 (d,
4JCF = 1.2 Hz, C), 137.4 (C), 136.3 (C), 133.5 (C), 130.1 (CH), 129.0 (C), 127.8 (CH),
125.8 (CH), 125.1 (CH), 116.3 (CH), 113.3 (CH), 91.4 (C), 82.8 (d,
1JCF = 204.8, CFH), 82.5 (C), 59.6 (d,
2JCF = 23.8 Hz, CH
2), 28.2 (CH
3), 0.17 (CH
3), -0.68 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
50-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 633 nm); MS (ESI) calcd for C
33H
35F
2N
2O
4Si [M+H]
+ 589.2, found 589.2.
Example 38. 2-(3,7-Bis(3-fluoroazetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-carboxybenzoate
[0222]

[0223] The procedure described for Example 19 was used to prepare the title compound from
Example 37 (80%, dark blue solid, TFA salt).
1H NMR (MeOD, 400 MHz) δ 8.25 (dd,
J = 8.0, 1.4 Hz, 1H), 8.11 (d,
J = 8.1 Hz, 1H), 7.84 (dd,
J = 1.4, 0.7 Hz, 1H), 6.87 (d,
J = 2.6 Hz, 2H), 6.83 (d,
J = 8.8 Hz, 2H), 6.39 (dd,
J = 8.9, 2.6 Hz, 2H), 5.43 (dtt,
2JHF = 57.3, 6.1, 3.3 Hz, 2H), 4.41 - 4.20 (m, 4H), 4.16 - 4.01 (m, 4H), 0.65 (s, 3H),
0.56 (s, 3H); Analytical HPLC: 88.1% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL
injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 633 nm); MS (ESI) calcd for C
29H
27F
2N
2O
4Si [M+H]
+ 533.2, found 533.0.
Example 39. 2-(3,7-Bis(3-fluoroazetidin-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-ylium-10(5H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate
[0224]

[0225] The procedure described for Example 35 was used to prepare the title compound from
Example 38 (61%, blue-green solid).
1H NMR (CDCl
3, 400 MHz) δ 7.99 (dd,
J= 7.9, 0.7 Hz, 1H), 7.89 (dd,
J= 8.0, 1.4 Hz, 1H), 7.69 (dd,
J= 1.4, 0.8 Hz, 1H), 6.81 (d,
J = 8.7 Hz, 2H), 6.78 (s, 1H), 6.69 (d,
J = 2.6 Hz, 2H), 6.36 - 6.26 (m, 2H), 5.41 (dtt,
2JHF = 56.9, 5.9, 3.7 Hz, 2H), 4.29 - 4.13 (m, 4H), 4.06 - 3.91 (m, 4H), 3.67 - 3.60 (m,
6H), 3.58 - 3.53 (m, 2H), 3.50 (t,
J = 6.6 Hz, 2H), 3.40 (t,
J = 6.7 Hz, 2H), 1.79 - 1.67 (m, 2H), 1.54 - 1.47 (m, 2H), 1.45 - 1.35 (m, 2H), 1.35
- 1.24 (m, 2H), 0.66 (s, 3H), 0.59 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -180.49 (dtt,
JFH = 56.9, 23.9, 18.2 Hz); Analytical HPLC: 98.7% purity (4.6 mm × 150 mm 5 µm C18 column;
5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 633 nm); MS (ESI) calcd for C
39H
47ClF
2N
3O
5Si [M+H]
+ 738.3, found 738.2.
Example 40. 2-(3,6-Di(azetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate
[0226]

[0227] The procedure described for Example 7 was used to prepare the title compound (88%,
pale blue solid) from carbofluorescein ditriflate (
Grimm, J. B.; Sung, A. J.; Legant, W. R.; Hulamm, P.; Matlosz, S. M.; Betzig, E.;
Lavis, L. D. ACS Chem. Biol. 2013, 8, 1303).
1H NMR (CDCl
3, 400 MHz) δ 8.00 - 7.95 (m, 1H), 7.58 (td,
J = 7.4, 1.4 Hz, 1H), 7.53 (td,
J = 7.4, 1.2 Hz, 1H), 7.08 - 7.03 (m, 1H), 6.58 (d,
J = 2.4 Hz, 2H), 6.55 (d,
J = 8.5 Hz, 2H), 6.20 (dd,
J = 8.6, 2.4 Hz, 2H), 3.90 (t,
J = 7.2 Hz, 8H), 2.37 (p,
J = 7.2 Hz, 4H), 1.82 (s, 3H), 1.72 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 170.9 (C), 155.6 (C), 152.4 (C), 146.9 (C), 134.5 (CH), 128.94 (CH),
128.89 (CH), 127.4 (C), 125.0 (CH), 124.1 (CH), 120.6 (C), 110.4 (CH), 107.9 (CH),
88.4 (C), 52.4 (CH
2), 38.6 (C), 35.7 (CH
3), 32.3 (CH
3), 17.0 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 600 nm); HRMS (ESI) calcd for C
29H
29N
2O
2 [M+H]
+ 437.2224, found 437.2236.
Example 41. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate
[0228]

[0229] The procedure described for Example 7 was used to prepare the title compound from
carbofluorescein ditriflate (
Grimm, J. B.; Sung, A. J.; Legant, W. R.; Hulamm, P.; Matlosz, S. M.; Betzig, E.;
Lavis, L. D. ACS Chem. Biol. 2013, 8, 1303) and 3,3-difluoroazetidine hydrochloride (95%, off-white solid).
1H NMR (CDCl
3, 400 MHz) δ 8.04 - 7.98 (m, 1H), 7.60 (td,
J = 7.3, 1.5 Hz, 1H), 7.56 (td,
J = 7.3, 1.3 Hz, 1H), 7.04 (s, 1H), 6.64 (d,
J = 2.8 Hz, 2H), 6.63 (d,
J = 8.7 Hz, 2H), 6.28 (dd,
J = 8.6, 2.5 Hz, 2H), 4.25 (t,
3JHF = 11.8 Hz, 8H), 1.84 (s, 3H), 1.74 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -99.95 (p,
3JFH = 11.8 Hz);
13C NMR (CDCl
3, 101 MHz) δ 170.6 (C), 155.2 (C), 150.1 (t,
4JCF = 2.7 Hz, C), 146.9 (C), 134.8 (CH), 129.3 (CH), 129.2 (CH), 127.0 (C), 125.2 (CH),
123.9 (CH), 122.4 (C), 115.9 (t,
1JCF = 274.6 Hz, CF
2), 111.6 (CH), 109.2 (CH), 87.2 (C), 63.4 (t,
2JCF = 25.9 Hz, CH
2), 38.6 (C), 35.6 (CH
3), 32.5 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
30-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 600 nm); MS (ESI) calcd for C
29H
25F
4N
2O
2 [M+H]
+ 509.2, found 509.1.
Example 42. 4-(tert-Butoxycarbonyl)-2-(3,6-di(-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate
[0230]

[0231] Step 1: A vial was charged with di-
tert-butyl 2-bromoterephthalate (Example 32, Step 1; 1.48 g, 4.14 mmol, 2 eq), sealed,
and flushed with nitrogen. After dissolving the bromide in THF (7 mL) and cooling
the reaction to -15 °C,
iPrMgCl·LiCl (1.3 M in THF, 3.19 mL, 4.14 mmol, 2 eq) was added. The reaction was warmed
to -10 °C and stirred for 4 h. A solution of 3,6-bis((
tert-butyldimethylsilyl)oxy)-10, 10-dimethylanthracen-9(10
H)-one (1.00 g, 2.07 mmol; from
Grimm, J. B.; Sung, A. J.; Legant, W. R.; Hulamm, P.; Matlosz, S. M.; Betzig, E.;
Lavis, L. D. ACS Chem. Biol. 2013, 8, 1303) in THF (4 mL) was then added dropwise. The reaction mixture was warmed to room temperature
and stirred for 2 h. It was subsequently quenched with saturated NH
4Cl, diluted with water, and extracted with EtOAc (2×). The combined organics were
washed with brine, dried (MgSO
4), filtered, and evaporated. Silica gel chromatography (0-10% Et
2O/hexanes, linear gradient) provided 245 mg (17%) of
tert-butyl 3,6-bis((
tertbutyldimethylsilyl)oxy)-10,10-dimethyl-3'-oxo-3'
H,10
H-spiro[anthracene-9,1'-isobenzofuran]-6'-carboxylate as a colorless solid.
1H NMR (CDCl
3, 400 MHz) δ 8.16 (dd,
J = 8.0, 1.3 Hz, 1H), 8.02 (dd,
J = 8.0, 0.6 Hz, 1H), 7.63 - 7.59 (m, 1H), 7.09 - 7.05 (m, 2H), 6.64 - 6.57 (m, 4H),
1.81 (s, 3H), 1.72 (s, 3H), 1.54 (s, 9H), 0.99 (s, 18H), 0.22 (s, 12H);
13C NMR (CDCl
3, 101 MHz) δ 169.9 (C), 164.4 (C), 156.5 (C), 155.5 (C), 147.0 (C), 138.1 (C), 130.3
(CH), 129.7 (C), 129.3 (CH), 125.1 (CH), 125.0 (CH), 124.0 (C), 119.2 (CH), 117.8
(CH), 87.0 (C), 82.5 (C), 38.2 (C), 35.0 (CH
3), 33.2 (CH
3), 28.2 (CH
3), 25.8 (CH
3), 18.4 (C), -4.17 (CH
3), -4.19 (CH
3); HRMS (ESI) calcd for C
40H
55O
6Si
2 [M+H]
+ 687.3537, found 687.3533.
[0232] Step 2: To a solution of the product from Step 1 (170 mg, 0.247 mmol) in THF (5 mL) was
added TBAF (1.0 M in THF, 990 µL, 0.990 mmol, 4 eq). The reaction was stirred at room
temperature for 10 min. It was subsequently diluted with saturated NH
4Cl and extracted with EtOAc (2×). The organic extracts were washed with brine, dried
(MgSO
4), filtered, and evaporated to provide an orange residue. The crude intermediate was
taken up in CH
2Cl
2 (5 mL) and cooled to 0 °C. Pyridine (160 µL, 1.98 mmol, 8 eq) and trifluoromethanesulfonic
anhydride (167 µL, 0.990 mmol, 4 eq) were added, and the ice bath was removed. The
reaction was stirred at room temperature for 2 h. It was then diluted with water and
extracted with CH
2Cl
2 (2×). The combined organics were washed with brine, dried (MgSO
4), filtered, and concentrated
in vacuo. Flash chromatography on silica gel (0-20% EtOAc/hexanes, linear gradient) afforded
159 mg (89%) of
tert-butyl 10,10-dimethyl-3'-oxo-3,6-bis(((trifluoromethyl)sulfonyl)oxy)-3'
H,10H-spiro[anthracene-9,1'-isobenzofuran]-6'-carboxylate as a colorless solid.
1H NMR (CDCl
3, 400 MHz) δ 8.24 (dd,
J = 8.0, 1.3 Hz, 1H), 8.11 (dd,
J = 8.0, 0.6Hz, 1H), 7.63 - 7.60 (m, 1H), 7.56 (d,
J = 2.5 Hz, 2H), 7.10 (dd,
J = 8.8, 2.5 Hz, 2H), 6.88 (d,
J = 8.8 Hz, 2H), 1.91 (s, 3H), 1.81 (s, 3H), 1.56 (s, 9H);
19F NMR (CDCl
3, 376 MHz) δ -73.20 (s);
13C NMR (CDCl
3, 101 MHz) δ 168.8 (C), 163.9 (C), 153.9 (C), 150.4 (C), 147.2 (C), 138.9 (C), 131.4
(CH), 131.1 (C), 130.3 (CH), 128.8 (C), 125.9 (CH), 124.7 (CH), 120.6 (CH), 119.8
(CH), 118.9 (q,
1JCF = 320.9 Hz, CF
3), 84.3 (C), 83.1 (C), 39.0 (C), 34.8 (CH
3), 33.2 (CH
3), 28.2 (CH
3); HRMS (ESI) calcd for C
30H
25F
6O
10S
2 [M+H]
+ 723.0793, found 723.0797.
[0233] Step 3: The procedure described for Example 7 was used to prepare the title compound 4-(
tert-butoxycarbonyl)-2-(3,6-di(azetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate
from the ditriflate synthesized in Step 2 (84%, blue solid).
1H NMR (CDCl
3, 400 MHz) δ 8.14 (dd,
J = 8.0, 1.3 Hz, 1H), 8.00 (dd,
J = 8.0, 0.7 Hz, 1H), 7.61 (dd,
J = 1.3, 0.8 Hz, 1H), 6.58 (d,
J = 2.3 Hz, 2H), 6.54 (d,
J = 8.6 Hz, 2H), 6.21 (dd,
J = 8.6, 2.4 Hz, 2H), 3.91 (t,
J = 7.2 Hz, 8H), 2.38 (p,
J = 7.2 Hz, 4H), 1.83 (s, 3H), 1.73 (s, 3H), 1.53 (s, 9H);
13C NMR (CDCl
3, 101 MHz) δ 170.1 (C), 164.6 (C), 155.6 (C), 152.4 (C), 146.8 (C), 137.8 (C), 130.3
(C), 130.1 (CH), 128.9 (CH), 125.1 (CH), 124.8 (CH), 119.9 (C), 110.5 (CH), 108.0
(CH), 88.8 (C), 82.3 (C), 52.3 (CH
2), 38.5 (C), 35.5 (CH
3), 32.8 (CH
3), 28.2 (CH
3), 17.0 (CH
2); HRMS (ESI) calcd for C
34H
37N
2O
4 [M+H]
+ 537.2753, found 537.2768.
Example 43. 4-Carboxy-2-(3,6-di(azetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate
[0234]

[0235] The procedure described for Example 19 was used to prepare the title compound from
Example 42 (98%, dark blue solid, TFA salt).
1H NMR (MeOD, 400 MHz) δ 8.34 (dd,
J = 8.2, 0.5 Hz, 1H), 8.31 (dd,
J = 8.2, 1.5 Hz, 1H), 7.84 (dd,
J= 1.5, 0.5 Hz, 1H), 6.93 (d,
J = 9.1 Hz, 2H), 6.82 (d,
J = 2.2 Hz, 2H), 6.39 (dd,
J = 9.1, 2.3 Hz, 2H), 4.33 (t,
J = 7.6 Hz, 8H), 2.55 (p,
J = 7.6 Hz, 4H), 1.82 (s, 3H), 1.70 (s, 3H);
19F NMR (MeOD, 376 MHz) δ -75.24 (s);
13C NMR (MeOD, 101 MHz) δ 167.9 (C), 167.5 (C), 165.4 (C), 158.0 (C), 156.8 (C), 139.3
(C), 137.6 (CH), 136.2 (C), 135.5 (C), 132.5 (CH), 132.4 (CH), 131.5 (CH), 121.8 (C),
111.9 (CH), 109.7 (CH), 52.9 (CH
2), 42.8 (C), 35.6 (CH
3), 32.0 (CH
3), 16.8 (CH
2); Analytical HPLC: >99% purity (4.6 mm x 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 600 nm); HRMS (ESI) calcd for C
30H
29N
2O
4 [M+H]
+ 481.2127, found 481.2120.
Example 44. 4-((2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)-2-(3,6-di(azetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)benzoate
[0236]

[0237] The procedure described for Example 35 was used to prepare the title compound from
Example 43 (72%, dark blue solid).
1H NMR (CDCl
3, 400 MHz) δ 8.02 (dd,
J = 8.0, 0.6 Hz, 1H), 7.94 (dd,
J = 8.0, 1.4 Hz, 1H), 7.42 - 7.40 (m, 1H), 6.68 - 6.62 (m, 1H), 6.57 (d,
J = 2.3 Hz, 2H), 6.52 (d,
J = 8.6 Hz, 2H), 6.20 (dd,
J = 8.6, 2.4 Hz, 2H), 3.91 (t,
J = 7.4 Hz, 8H), 3.64 - 3.56 (m, 6H), 3.55 - 3.48 (m, 4H), 3.38 (t
, J = 6.6 Hz, 2H), 2.37 (p,
J = 7.2 Hz, 4H), 1.83 (s, 3H), 1.77 - 1.68 (m, 2H), 1.72 (s, 3H), 1.56 - 1.47 (m, 2H),
1.46 - 1.36 (m, 2H), 1.36 - 1.28 (m, 2H); Analytical HPLC: >99% purity (4.6 mm x 150
mm 5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; UV detection at 600 nm); HRMS (ESI) calcd for C
40H
49ClN
3O
5 [M+H]
+ 686.3361, found 686.3375.
Example 45. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-(tert-butoxycarbonyl)benzoate
[0238]

[0239] The procedure described for Example 1 was used to prepare the title compound from
tert-butyl 10,10-dimethyl-3'-oxo-3,6-bis(((trifluoromethyl)sulfonyl)oxy)-3'
H,10
H-spiro[anthracene-9,1'-isobenzofuran]-6'-carboxylate (Example 42, Step 2) and 3,3-difluoroazetidine
hydrochloride (93%, off-white solid).
1H NMR (CDCl
3, 400 MHz) δ 8.16 (dd,
J = 8.0, 1.3 Hz, 1H), 8.02 (dd,
J = 8.0, 0.7 Hz, 1H), 7.60 (dd,
J = 1.2, 0.8 Hz, 1H), 6.65 (d,
J = 2.4 Hz, 2H), 6.62 (d,
J = 8.6 Hz, 2H), 6.29 (dd,
J = 8.6, 2.5 Hz, 2H), 4.26 (t,
3JHF = 11.7 Hz, 8H), 1.85 (s, 3H), 1.75 (s, 3H), 1.53 (s, 9H);
19F NMR (CDCl
3, 376 MHz) δ - 99.96 (p,
3JFH = 11.8 Hz);
13C NMR (CDCl
3, 101 MHz) δ 169.9 (C), 164.4 (C), 155.3 (C), 150.1 (t,
4JCF = 2.7 Hz, C), 146.8 (C), 138.1 (C), 130.3 (CH), 129.9 (C), 129.2 (CH), 125.1 (CH),
124.9 (CH), 121.7 (C), 115.9 (t,
1JCF = 274.6 Hz, CF
2), 111.8 (CH), 109.3 (CH), 87.5 (C), 82.5 (C), 63.4 (t,
2JCF = 26.0 Hz, CH
2), 38.5 (C), 35.4 (CH
3), 33.0 (CH
3), 28.2 (CH
3); MS (ESI) calcd for C
34H
33F
4N
2O
4 [M+H]
+ 609.2, found 609.3.
Example 46. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-carboxybenzoate
[0240]

[0241] The procedure described for Example 19 was used to prepare the title compound from
Example 45 (99%, dark blue-purple solid, TFA salt).
1H NMR (MeOD, 400 MHz) δ 8.30 (dd,
J = 8.1, 1.4 Hz, 1H), 8.23 - 8.15 (m, 1H), 7.69 (s, 1H), 6.92 (d,
J = 2.1 Hz, 2H), 6.79 (d,
J = 7.6 Hz, 2H), 6.47 (dd,
J = 8.8, 2.3 Hz, 2H), 4.45 (t,
3JHF = 11.0 Hz, 8H), 1.88 (s, 3H), 1.76 (s, 3H);
19F NMR (MeOD, 376 MHz) δ - 75.81 (s, 3F), -100.32 (m, 4F); Analytical HPLC: >99% purity
(4.6 mm × 150 mm 5 µm C18 column; 5 µL injection; 30-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 600 nm); MS (ESI) calcd for C
30H
25F
4N
2O
4 [M+H]
+ 553.2, found 553.1.
Example 47. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate
[0242]

[0243] The procedure described for Example 34 was used to prepare the title compound from
Example 46 (93%, yellow solid).
1H NMR (CDCl
3, 400 MHz) δ 8.32 (dd,
J= 8.0, 1.4 Hz, 1H), 8.14 (dd,
J = 8.0, 0.7 Hz, 1H), 7.74 (dd,
J= 1.3, 0.8 Hz, 1H), 6.65 (d,
J= 2.4 Hz, 2H), 6.58 (d,
J= 8.6 Hz, 2H), 6.31 (dd,
J= 8.6, 2.5 Hz, 2H), 4.26 (t,
3JHF = 11.7 Hz, 8H), 2.88 (s, 4H), 1.84 (s, 3H), 1.73 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -99.97 (p,
JFH = 11.7 Hz);
13C NMR (CDCl
3, 101 MHz) δ 169.1 (C), 168.9 (C), 160.9 (C), 155.6 (C), 150.3 (t,
4JCF = 2.5 Hz, C), 146.9 (C), 132.0 (C), 131.3 (CH), 131.0 (C), 129.3 (CH), 126.2 (CH),
125.8 (CH), 120.9 (C), 115.9 (t,
1JCF = 274.5 Hz, CF
2), 112.0 (CH), 109.4 (CH), 87.9 (C), 63.4 (t,
2JCF = 26.0 Hz, CH
2), 38.5 (C), 35.5 (CH
3), 32.8 (CH
3), 25.8 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
30-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 600 nm); MS (ESI) calcd for C
34H
28F
4N
3O
6 [M+H]
+ 650.2, found 650.1.
Example 48. 2-(3,6-Bis(3,3-difluoroazetidin-1-yl)-10,10-dimethylanthracen-9-ylium-9(10H)-yl)-4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate
[0244]

[0245] The procedure described for Example 35 was used to prepare the title compound from
Example 46 (54%, off-white/bluish solid).
1H NMR (CDCl
3, 400 MHz) δ 8.04 (dd,
J = 8.0, 0.5 Hz, 1H), 7.93 (dd,
J = 8.0, 1.4 Hz, 1H), 7.44 - 7.40 (m, 1H), 6.70 - 6.65 (m, 1H), 6.64 (d,
J = 2.4 Hz, 2H), 6.60 (d,
J = 8.6 Hz, 2H), 6.28 (dd,
J = 8.6, 2.4 Hz, 2H), 4.25 (t,
3JHF = 11.7 Hz, 8H), 3.66 - 3.57 (m, 6H), 3.56 - 3.48 (m, 4H), 3.39 (t,
J = 6.6 Hz, 2H), 1.85 (s, 3H), 1.79 - 1.70 (m, 5H), 1.57 - 1.48 (m, 2H), 1.46 - 1.37
(m, 2H), 1.37 - 1.26 (m, 2H);
19F NMR (CDCl
3, 376 MHz) δ -99.94 (p,
3JFH = 11.8 Hz); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
30-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 600 nm); MS (ESI) calcd for C
40H
45ClF
4N
3O
5 [M+H]
+ 758.3, found 758.2.
Example 49. 7-(Azetidin-1-yl)-4-methyl-2H-chromen-2-one
[0246]

[0247] A vial was charged with 4-methylumbelliferone triflate (300 mg, 0.973 mmol; from
Kövér, J.; Antus, S. Z. Naturforsch., B: J. Chem. Sci. 2005, 60, 792), RuPhos-G3-palladacycle
(41 mg, 0.049 mmol, 0.05 eq), RuPhos (23 mg, 0.049 mmol, 0.05 eq), and K
2CO
3 (188 mg, 1.36 mmol, 1.4 eq). The vial was sealed and evacuated/backfilled with nitrogen
(3×). Dioxane (8 mL) was added, and the reaction was flushed again with nitrogen (3×).
Following the addition of azetidine (72 µL, 1.07 mmol, 1.1 eq), the reaction was stirred
at 80 °C for 6.5 h. It was then cooled to room temperature, deposited onto Celite,
and concentrated to dryness. Purification by silica gel chromatography (0-30% EtOAc/hexanes,
linear gradient; dry load with Celite) afforded the title compound (190 mg, 91%) as
a yellow solid.
1H NMR (CDCl
3, 400 MHz) δ 7.38 (d,
J = 8.6 Hz, 1H), 6.30 (dd,
J = 8.6, 2.3 Hz, 1H), 6.22 (d,
J = 2.3 Hz, 1H), 5.97 (q,
J = 1.1 Hz, 1H), 4.03 - 3.95 (m, 4H), 2.44 (p,
J = 7.3 Hz, 2H), 2.34
(d, J = 1.1 Hz, 3H);
13C NMR (CDCl
3, 101 MHz) δ 162.0 (C), 155.7 (C), 154.0 (C), 153.1 (C), 125.5 (CH), 110.4 (C), 109.5
(CH), 107.8 (CH), 97.2 (CH), 51.9 (CH
2), 18.7 (CH
3), 16.6 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 350 nm); HRMS (ESI) calcd for C
13H
14NO
2 [M+H]
+ 216.1019, found 216.1014.
Example 50. Ethyl 7-(azetidin-1-yl)-2-oxo-2H-chromene-3-carboxylate
[0248]

[0249] Step 1: A flask was charged with Pd(OAc)
2 (130 mg, 0.578 mmol, 0.05 eq), sealed, and evacuated/backfilled with nitrogen (3×).
Toluene (40 mL) was added; separate solutions of 3-bromophenol (2.00 g, 11.6 mmol)
in toluene (8 mL), 2,8,9-triisobutyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane
("Verkade base," 396 mg, 1.16 mmol, 0.1 eq) in toluene (8 mL), and LiHMDS (1.0 M in
THF, 26.6 mL, 26.6 mmol, 2.3 eq) were then added sequentially. Following the addition
of azetidine (935 µL, 13.9 mmol, 1.2 eq), the reaction was stirred at 80 °C for 18
h. It was then cooled to room temperature, deposited onto Celite, and concentrated
to dryness. Purification by silica gel chromatography (0-35% EtOAc/hexanes, linear
gradient; dry load with Celite) afforded 3-(azetidin-1-yl)phenol (1.44 g, 84%) as
an off-white solid.
1H NMR (CDCl
3, 400 MHz) δ 7.05 (t,
J= 8.0 Hz, 1H), 6.19 (ddd,
J = 8.0, 2.4, 0.8 Hz, 1H), 6.04 (ddd,
J = 8.1, 2.1, 0.8 Hz, 1H), 5.92 (t,
J = 2.3 Hz, 1H), 4.77 (s, 1H), 3.89 - 3.81 (m, 4H), 2.34 (p,
J = 7.2 Hz, 2H);
13C NMR (CDCl
3, 101 MHz) δ 156.7 (C), 153.9 (C), 130.1 (CH), 105.1 (CH), 104.5 (CH), 99.0 (CH),
52.7 (CH
2), 17.0 (CH
2); HRMS (ESI) calcd for C
9H
12NO [M+H]
+ 150.0913, found 150.0915.
[0250] Step 2: DMF (2 mL) was cooled to 0 °C under nitrogen, and POCl
3 (500 µL, 5.36 mmol, 2 eq) was added dropwise. The ice bath was then removed, and
the reaction was stirred at room temperature for 1 h. The phenol from Step 1 (400
mg, 2.68 mmol) in DMF (4 mL) was then added. After stirring the reaction at room temperature
for 1 h, it was carefully diluted with saturated NaHCO
3 (∼20 mL) and EtOAc (∼20 mL) and vigorously stirred for 10 min. The mixture was diluted
with additional water and extracted with EtOAc (2×). The combined organic extracts
were washed with water and brine, dried (MgSO
4), filtered, and concentrated
in vacuo. The residue was purified by flash chromatography on silica gel (0-40% EtOAc/hexanes,
linear gradient) to yield 230 mg (48%) of 4-(azetidin-1-yl)-2-hydroxybenzaldehyde
as a white solid.
1H NMR (CDCl
3, 400 MHz) δ 11.69 (s, 1H), 9.50 (s, 1H), 7.25 (d,
J= 8.5 Hz, 1H), 5.94 (dd,
J = 8.5, 2.1 Hz, 1H), 5.75 (d,
J = 2.1 Hz, 1H), 4.08 - 3.98 (m, 4H), 2.43 (p,
J = 7.4 Hz, 2H);
13C NMR (CDCl
3, 101 MHz) δ 192.6 (CH), 164.4 (C), 156.6 (C), 135.5 (CH), 112.2 (C), 103.2 (CH),
95.6 (CH), 51.2 (CH
2), 16.3 (CH
2); HRMS (ESI) calcd for C
10H
12NO
2 [M+H]
+ 178.0863, found 178.0866.
[0251] Step 3: The aldehyde from Step 2 (175 mg, 0.988 mmol) was suspended in EtOH (10 mL). Diethyl
malonate (301 µL, 1.98 mmol, 2 eq) and piperidine (29 µL, 0.296 mmol, 0.3 eq) were
added, and the reaction was stirred at reflux for 12 h. It was then cooled to room
temperature and allowed to stand for 12 h, during which time a yellow solid crystallized
out of the solution. The mixture were filtered; the filter cake was washed with EtOH
and dried to afford 232 mg (86%) of the title compound ethyl 7-(azetidin-1-yl)-2-oxo-2
H-chromene-3-carboxylate as a bright yellow crystalline solid.
1H NMR (CDCl
3, 400 MHz) δ 8.39 (s, 1H), 7.31 (d,
J = 8.6 Hz, 1H), 6.26 (dd,
J = 8.6, 2.2 Hz, 1H), 6.09 (d,
J = 2.1 Hz, 1H), 4.37 (q,
J = 7.1 Hz, 2H), 4.10 - 4.02 (m, 4H), 2.48 (p,
J = 7.4 Hz, 2H), 1.38 (t,
J = 7.1 Hz, 3H);
13C NMR (CDCl
3, 101 MHz) δ 164.2 (C), 158.13 (C), 158.12 (C), 155.2 (C), 149.5 (CH), 131.0 (CH),
109.4 (C), 108.39 (C), 108.36 (CH), 95.4 (CH), 61.2 (CH
2), 51.4 (CH
2), 16.3 (CH
2), 14.5 (CH
3); HRMS (ESI) calcd for C
15H
15NO
4Na [M+Na]
+ 296.0893, found 296.0900.
Example 51. 7-(Azetidin-1-yl)-2-oxo-2H-chromene-3-carboxylic acid
[0252]

[0253] Ethyl 7-(azetidin-1-yl)-2-oxo-2
H-chromene-3-carboxylate (Example 50; 65 mg, 0.238 mmol) was taken up in 1:1 THF/MeOH
(8 mL), and 1 M NaOH (476 µL, 0.476 mmol, 2 eq) was added. The reaction was stirred
at room temperature for 3 h. It was then acidified with 1 M HCl (500 µL), and the
resulting yellow suspension was filtered. The filter cake was washed (water, EtOAc)
and dried to provide the title compound (47 mg, 81%) as a bright yellow solid.
1H NMR (DMSO-d
6, 400 MHz) δ 12.52 (s, 1H), 8.59 (s, 1H), 7.64 (d,
J = 8.7 Hz, 1H), 6.42 (dd,
J = 8.7, 2.2 Hz, 1H), 6.23 (d,
J = 1.9 Hz, 1H), 4.10 - 4.01 (m, 4H), 2.39 (p,
J = 7.4 Hz, 2H);
13C NMR (DMSO-d
6, 101 MHz) δ 164.4 (C), 159.1 (C), 157.4 (C), 155.1 (C), 149.7 (CH), 131.7 (CH), 108.7
(CH), 108.0 (C), 107.6 (C), 94.7 (CH), 51.2 (CH
2), 15.6 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 400 nm); HRMS (ESI) calcd for C
13H
12NO
4 [M+H]
+ 246.0761, found 246.0770.
Example 52. N-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-7-(azetidin-1-yl)-2-oxo-2H-chromene-3-carboxamide
[0254]

[0255] 7-(Azetidin-1-yl)-2-oxo-2
H-chromene-3-carboxylic acid (Example 51; 6.0 mg, 24.5 µmol) was combined with TSTU
(11.0 mg, 36.7 µmol, 1.5 eq) in DMF (1 mL). After adding DIEA (21.3 µL, 122 µmol,
5 eq), the reaction was stirred at room temperature for 1 h while shielded from light.
6-((4-(Aminomethyl)benzyl)oxy)-9
H-purin-2-amine ("BG-NH
2," 9.9 mg, 36.7 µmol, 1.5 eq) was then added. The reaction was stirred an additional
2 h at room temperature. Purification of the crude reaction mixture by reverse phase
HPLC (10-75% MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) afforded 11.5 mg (77%, TFA
salt) of the title compound as a yellow solid.
1H NMR (MeOD, 400 MHz) δ 8.65 (s, 1H), 8.31 (s, 1H), 7.57 - 7.50 (m, 3H), 7.41 (d,
J = 8.1 Hz, 2H), 6.45 (dd,
J = 8.7, 2.1 Hz, 1H), 6.22 (d,
J = 2.0 Hz, 1H), 5.64 (s, 2H), 4.62 (s, 2H), 4.15 - 4.06 (m, 4H), 2.48 (p,
J = 7.4 Hz, 2H);
19F NMR (MeOD, 376 MHz) δ -75.44 (s); Analytical HPLC: >99% purity (4.6 mm × 150 mm
5 µm C18 column; 5 µL injection; 10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 400 nm); HRMS (ESI) calcd for C
26H
24N
7O
4 [M+H]
+ 498.1884, found 498.1891.
Example 53. Methyl 2-(7-(azetidin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)acetate
[0256]

[0257] Step 1: Methyl 7-hydroxy-4-methylcoumarin-3-acetate (1.00 g, 4.03 mmol; from
Franzini, R. M.; Kool, E. T. Chembiochem 2008, 9, 2981),
N-phenyl-bis(trifluoromethanesulfonimide) (1.58 g, 4.43 mmol, 1.1 eq), and DIEA (912
µL, 5.24 mmol, 1.3 eq) were combined in MeCN (20 mL) and stirred at room temperature
for 18 h. The reaction mixture was concentrated to dryness, and the resulting residue
was purified by flash chromatography on silica gel (0-50% EtOAc/hexanes, linear gradient)
to afford methyl 2-(4-methyl-2-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-2
H-chromen-3-yl)acetate (1.41 g, 92%) as an off-white solid.
1H NMR (CDCl
3, 400 MHz) δ 7.73 (d,
J = 8.8 Hz, 1H), 7.29 (d,
J = 2.4 Hz, 1H), 7.26 (dd,
J = 8.8, 2.5 Hz, 1H), 3.76 (s, 2H), 3.73 (s, 3H), 2.44 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -73.08 (s);
13C NMR (CDCl
3, 101 MHz) δ 170.2 (C), 160.4 (C), 153.0 (C), 150.5 (C), 147.9 (C), 126.7 (CH), 121.0
(C), 120.5 (C), 118.8 (q,
1JCF = 321.0 Hz, CF
3), 117.6 (CH), 110.5 (CH), 52.6 (CH
3), 33.0 (CH
2), 15.7 (CH
3); HRMS (ESI) calcd for C
14H
11F
3O
7SNa [M+Na]
+ 403.0070, found 403.0081.
[0258] Step 2: A vial was charged with the triflate from Step 1 (300 mg, 0.789 mmol), RuPhos-G3-palladacycle
(33 mg, 0.039 mmol, 0.05 eq), RuPhos (18 mg, 0.039 mmol, 0.05 eq), and K
2CO
3 (153 mg, 1.10 mmol, 1.4 eq). The vial was sealed and evacuated/backfilled with nitrogen
(3×). Dioxane (5 mL) was added, and the reaction was flushed again with nitrogen (3×).
Following the addition of azetidine (58 µL, 0.868 mmol, 1.1 eq), the reaction was
stirred at 80 °C for 4 h. It was then cooled to room temperature, diluted with CH
2Cl
2, deposited onto Celite, and concentrated to dryness. Purification by silica gel chromatography
(0-50% EtOAc/hexanes, linear gradient, with constant 40% v/v CH
2Cl
2; dry load with Celite) afforded the title compound methyl 2-(7-(azetidin-1-yl)-4-methyl-2-oxo-2
H-chromen-3-yl)acetate (210 mg, 93%) as a pale yellow solid.
1H NMR (CDCl
3, 400 MHz) δ 7.42 (d,
J = 8.7 Hz, 1H), 6.32 (dd,
J = 8.7, 2.3 Hz, 1H), 6.22 (d,
J = 2.3 Hz, 1H), 4.03 - 3.94 (m, 4H), 3.70 (s, 3H), 3.69 (s, 2H), 2.43 (p,
J = 7.3 Hz, 2H), 2.33 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 171.4 (C), 162.4 (C), 154.6 (C), 153.7 (C), 149.8 (C), 125.7 (CH), 113.7
(C), 110.7 (C), 108.0 (CH), 97.0 (CH), 52.2 (CH
3), 51.9 (CH
2), 32.8 (CH
2), 16.6 (CH
2), 15.3 (CH
3); HRMS (ESI) calcd for C
16H
17NO
4Na [M+Na]
+ 310.1050, found 310.1068.
Example 54. Methyl 2-(7-(3,3-difluoroazetidin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)acetate
[0259]

[0260] A vial was charged with methyl 2-(4-methyl-2-oxo-7-(((trifluoromethyl)sulfonyl)oxy)-2
H-chromen-3-yl)acetate (Example 53, Step 1; 240 mg, 0.631 mmol), RuPhos-G3-palladacycle
(26 mg, 0.032 mmol, 0.05 eq), RuPhos (15 mg, 0.032 mmol, 0.05 eq), K
2CO
3 (218 mg, 1.58 mmol, 2.5 eq), and 3,3-difluoroazetidine hydrochloride (84 mg, 0.694
mmol, 1.1 eq). The vial was sealed and evacuated/backfilled with nitrogen (3×). Dioxane
(4 mL) was added, and the reaction was flushed again with nitrogen (3×). The mixture
was then stirred at 80 °C for 24 h. It was subsequently cooled to room temperature,
diluted with CH
2Cl
2, deposited onto Celite, and concentrated to dryness. Purification by silica gel chromatography
(0-40% EtOAc/hexanes, linear gradient, with constant 40% v/v CH
2Cl
2; dry load with Celite) afforded the title compound as an off-white solid (158 mg,
77%).
1H NMR (CDCl
3, 400 MHz) δ 7.50 (d,
J = 8.7 Hz, 1H), 6.41 (dd,
J = 8.7, 2.4 Hz, 1H), 6.34 (d,
J = 2.4 Hz, 1H), 4.32 (t,
3JHF = 11.7 Hz, 4H), 3.71 (s, 3H), 3.71 (s, 2H), 2.36 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -100.14 (p,
3JFH = 11.6 Hz);
13C NMR (CDCl
3, 101 MHz) δ 171.1 (C), 161.9 (C), 154.3 (C), 151.5 (t,
4JCF = 3.2 Hz, C), 149.4 (C), 126.1 (CH), 115.5 (t,
1JCF = 274.6 Hz, CF
2), 115.4 (C), 112.3 (C), 109.0 (CH), 98.9 (CH), 63.3 (t,
2JCF = 26.8 Hz, CH
2), 52.3 (CH
3), 32.8 (CH
2), 15.4 (CH
3); HRMS (ESI) calcd for C
16H
15F
2NO
4Na [M+Na]
+ 346.0861, found 346.0872.
Example 55. 2-(7-(Azetidin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)acetic acid
[0261]

[0262] Methyl 2-(7-(azetidin-1-yl)-4-methyl-2-oxo-2
H-chromen-3-yl)acetate (Example 53; 190 mg, 0.661 mmol) was dissolved in 1:1 THF/MeOH
(8 mL), and 1 M NaOH (1.32 mL, 1.32 mmol, 2 eq) was added. After stirring the reaction
at room temperature for 24 h, it was acidified with 1 M HCl (1.40 mL), diluted with
water, and extracted with EtOAc (2×). The combined organic extracts were washed with
brine, dried (MgSO
4), filtered, and concentrated
in vacuo. The resulting residue was triturated with CH
2Cl
2/hexanes, filtered, and dried to provide 164 mg (91%) of the title compound as a pale
yellow solid.
1H NMR (DMSO-d
6, 400 MHz) δ 12.33 (s, 1H), 7.58 (d,
J = 8.8 Hz, 1H), 6.40 (dd,
J = 8.7, 2.3 Hz, 1H), 6.25 (d,
J = 2.3 Hz, 1H), 3.94 (t,
J = 7.3 Hz, 4H), 3.52 (s, 2H), 2.36 (p,
J = 7.3 Hz, 2H), 2.30 (s, 3H);
13C NMR (DMSO-d
6, 101 MHz) δ 171.8 (C), 161.2 (C), 153.7 (C), 153.4 (C), 149.5 (C), 126.2 (CH), 113.6
(C), 109.7 (C), 108.0 (CH), 96.1 (CH), 51.5 (CH
2), 32.5 (CH
2), 16.0 (CH
2), 14.9 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 350 nm); HRMS (ESI) calcd for C
15H
15NO
4Na [M+Na]
+ 296.0893, found 296.0909.
Example 56. 2-(7-(3,3-Difluoroazetidin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)acetic acid
[0263]

[0264] The procedure described for Example 55 was used to prepare the title compound from
Example 54 (93%, white solid).
1H NMR (DMSO-d
6, 400 MHz) δ 12.37 (s, 1H), 7.67 (d,
J = 8.7 Hz, 1H), 6.58 (dd
, J = 8.7, 2.4 Hz, 1H), 6.51 (d
, J = 2.3 Hz, 1H), 4.42 (t,
3JHF = 12.3 Hz, 4H), 3.55 (s, 2H), 2.33 (s, 3H);
19F NMR (DMSO-d
6, 376 MHz) δ -98.45 (p,
3JHF = 12.4 Hz);
13C NMR (DMSO-d
6, 101 MHz) δ 171.7 (C), 161.0 (C), 153.3 (C), 151.7 (t,
4JCF = 3.2 Hz, C), 149.3 (C), 126.4 (CH), 116.4 (t,
1JCF = 272.9 Hz, CF
2), 115.1 (C), 111.3 (C), 109.6 (CH), 98.6 (CH), 62.8 (t,
2JCF = 26.1 Hz, CH
2), 32.6 (CH
2), 14.9 (CH
3); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 350 nm); HRMS (ESI) calcd for C
15H
13F
2NO
4Na [M+Na]
+ 332.0705, found 332.0714.
Example 57. 2,5-Dioxopyrrolidin-1-yl 2-(7-(azetidin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)acetate
[0265]

[0266] To a solution of 2-(7-(azetidin-1-yl)-4-methyl-2-oxo-2
H-chromen-3-yl)acetic acid (Example 55; 75 mg, 0.274 mmol) and TSTU (124 mg, 0.412
mmol, 1.5 eq) in DMF (4 mL) was added DIEA (96 µL, 0.549 mmol, 2 eq). The reaction
was stirred at room temperature for 4 h. It was subsequently diluted with 10% w/v
citric acid and extracted with EtOAc (2x). The combined organic extracts were washed
with water and brine, dried (MgSO
4), filtered, and concentrated
in vacuo. Silica gel chromatography (0-50% EtOAc/CH
2Cl
2, linear gradient) yielded 84 mg (83%) of the title compound as a pale yellow solid.
1H NMR (CDCl
3, 400 MHz) δ 7.43 (d,
J = 8.7 Hz, 1H), 6.31 (dd,
J = 8.7, 2.3 Hz, 1H), 6.21 (d,
J = 2.3 Hz, 1H), 4.02 (s, 2H), 4.02 - 3.96 (m, 4H), 2.81 (s, 4H), 2.44 (p,
J = 7.3 Hz, 2H), 2.38 (s, 3H);
13C NMR (CDCl
3, 101 MHz) δ 169.0 (C), 166.4 (C), 162.0 (C), 154.7 (C), 154.0 (C), 151.3 (C), 125.9
(CH), 111.2 (C), 110.3 (C), 108.1 (CH), 96.9 (CH), 51.8 (CH
2), 29.8 (CH
2), 25.7 (CH
2), 16.6 (CH
2), 15.5 (CH
3); HRMS (ESI) calcd for C
19H
18N
2O
6Na [M+Na]
+ 393.1057, found 393.1065.
Example 58. 2,5-Dioxopyrrolidin-1-yl 2-(7-(3,3-difluoroazetidin-1-yl)-4-methyl-2-oxo-2H-chromen-3-yl)acetate
[0267]

[0268] The procedure described for Example 57 was used to prepare the title compound from
Example 56 (82%, white solid).
1H NMR (DMSO-d
6, 400 MHz) δ 7.71 (d,
J = 8.8 Hz, 1H), 6.59 (dd,
J = 8.7, 2.4 Hz, 1H), 6.53 (d,
J = 2.3 Hz, 1H), 4.43 (t,
3JHF = 12.3 Hz, 4H), 4.03 (s, 2H), 2.79 (s, 4H), 2.40 (s, 3H);
19F NMR (DMSO-d
6, 376 MHz) δ -98.49 (p,
3JFH = 12.3 Hz);
13C NMR (DMSO-d
6, 101 MHz) δ 170.0 (C), 166.5 (C), 160.6 (C), 153.5 (C), 152.1 (t,
4JCF = 3.2 Hz, C), 151.2 (C), 126.7 (CH), 116.4 (t,
1JCF = 272.8 Hz, CF
2), 112.1 (C), 110.9 (C), 109.8 (CH), 98.5 (CH), 62.8 (t,
2JCF = 26.2 Hz, CH
2), 29.5 (CH
2), 25.4 (CH
2), 15.1 (CH
3); HRMS (ESI) calcd for C
19H
16F
2N
2O
6Na [M+Na]
+ 429.0869, found 429.0876.
Example 59. 2-(6-(Azetidin-1-yl)-3-oxo-3H-xanthen-9-yl)benzoic acid
[0269]

[0270] Step 1: A vial was charged with fluorescein ditriflate (500 mg, 0.838 mmol), Pd
2dba
3 (38 mg, 0.042 mmol, 0.05 eq), XPhos (60 mg, 0.126 mmol, 0.15 eq), and Cs
2CO
3 (382 mg, 1.17 mmol, 1.4 eq). The vial was sealed and evacuated/backfilled with nitrogen
(3×). Dioxane (4 mL) was added, and the reaction was flushed again with nitrogen (3×).
Following the addition of azetidine (57 µL, 0.838 mmol, 1 eq), the reaction was stirred
at 80 °C for 2 h. It was then cooled to room temperature, deposited onto Celite, and
concentrated to dryness. Purification by silica gel chromatography (0-35% EtOAc/hexanes,
linear gradient; dry load with Celite) afforded 3'-(azetidin-1-yl)-3-oxo-3
H-spiro[isobenzofuran-1,9'-xanthen]-6'-yl trifluoromethanesulfonate (125 mg, 30%) as
an off-white solid. MS (ESI) calcd for C
24H
17F
3NO
6S [M+H]
+ 504.1, found 504.2.
[0271] Step 2: The product of Step 1 (72 mg, 0.143 mmol) was taken up in 1:1 THF/MeOH (5 mL), and
1 M NaOH (286 µL, 0.286 mmol, 2 eq) was added. After stirring the reaction at room
temperature for 6 h, the reaction was concentrated to dryness. The residue was purified
by reverse phase HPLC (10-95% MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) to yield 40 mg (58%) of the
title compound 2-(6-(azetidin-1-yl)-3-oxo-3
H-xanthen-9-yl)benzoic acid as a bright orange solid.
1H NMR (MeOD, 400 MHz) δ 8.36 - 8.30 (m, 1H), 7.85 (td,
J = 7.5, 1.5 Hz, 1H), 7.81 (td,
J = 7.6, 1.5 Hz, 1H), 7.43 - 7.38 (m, 1H), 7.16 (d,
J = 9.3 Hz, 1H), 7.15 (d,
J = 9.0 Hz, 1H), 7.08 (d,
J = 2.3 Hz, 1H), 6.93 (dd,
J = 9.0, 2.3 Hz, 1H), 6.74 (dd,
J = 9.3, 2.2 Hz, 1H), 6.64 (d,
J = 2.2 Hz, 1H), 4.44 - 4.35 (m, 4H), 2.58 (p,
J = 7.7 Hz, 2H);
13C NMR (MeOD, 101 MHz) δ 168.4 (C), 168.0 (C), 161.0 (C), 159.9 (C), 159.1 (C), 157.9
(C), 135.7 (C), 134.0 (CH), 133.1 (CH), 132.42 (CH), 132.40 (CH), 132.1 (C), 131.7
(CH), 131.2 (CH), 118.0 (CH), 117.1 (C), 116.4 (C), 115.9 (CH), 103.3 (CH), 95.1 (CH),
53.4 (CH
2), 16.7 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 500 nm); HRMS (ESI) calcd for C
23H
18NO
4 [M+H]
+ 372.1230, found 372.1230.
Example 60 (not according to the invention). 3,6-Di(azetidin-1-yl)acridine
[0272]

[0273] Step 1: Proflavine hydrochloride (250 mg, 1.02 mmol) was suspended in water (1 mL) in a microwave
vial, and concentrated H
2SO
4 (450 µL) was added. The sealed mixture was heated in a microwave at 195 °C for 8
h. The brown suspension was diluted with water and filtered; the resulting filter
cake was washed with water and dried to provide crude 3,6-dihydroxyacridine as a red-brown
solid (260 mg). The 3,6-dihydroxyacridine (260 mg, 1.23 mmol) was then suspended in
CH
2Cl
2 (5 mL). Pyridine (796 µL, 9.85 mmol, 8 eq) and trifluoromethanesulfonic anhydride
(828 µL, 4.92 mmol, 4 eq) were added, and the reaction was stirred at room temperature
for 2 h. It was subsequently diluted with water and extracted with CH
2Cl
2 (2×). The combined organic extracts were dried (MgSO
4), filtered, and concentrated
in vacuo. Purification by flash chromatography on silica gel (0-30% EtOAc/hexanes, linear gradient)
afforded 303 mg (63%, 2 steps) of acridine-3,6-diyl bis(trifluoromethanesulfonate)
as an off-white solid.
1H NMR (CDCl
3, 400 MHz) δ 8.93 (s, 1H), 8.19 - 8.13 (m, 4H), 7.54 (dd,
J = 9.2, 2.4 Hz, 2H);
19F NMR (CDCl
3, 376 MHz) δ -73.10 (s);
13C NMR (CDCl
3, 101 MHz) δ 151.1 (C), 149.5 (C), 137.0 (CH), 131.2 (CH), 125.7 (C), 121.4 (CH),
120.8 (CH), 119.0 (q,
1JCF = 321.0 Hz, CF
3); HRMS (ESI) calcd for C
15H
8F
6NO
6S
2 [M+H]
+ 475.9692, found 475.9689.
[0274] Step 2: A vial was charged with the ditriflate from Step 1 (200 mg, 0.421 mmol), Pd(OAc)
2 (19 mg, 0.084 mmol, 0.2 eq), BINAP (79 mg, 0.126 mmol, 0.3 eq), and Cs
2CO
3 (384 mg, 1.18 mmol, 2.8 eq). The vial was sealed and evacuated/backfilled with nitrogen
(3×). Toluene (2.5 mL) was added, and the reaction was flushed again with nitrogen
(3×). Following the addition of azetidine (68 µL, 1.01 mmol, 2.4 eq), the reaction
was stirred at 100 °C for 18 h. It was then cooled to room temperature, diluted with
MeOH, deposited onto Celite, and concentrated to dryness. Purification by silica gel
chromatography (0-10% MeOH (2 M NH
3)/CH
2Cl
2, linear gradient; dry load with Celite) afforded the title compound 3,6-di(azetidin-1-yl)acridine
(89 mg, 73%) as a red-orange solid.
1H NMR (MeOD, 400 MHz) δ 8.44 (s, 1H), 7.74 (d,
J = 9.0 Hz, 2H), 6.78 (dd,
J = 9.0, 2.2 Hz, 2H), 6.57 (d,
J = 2.0 Hz, 2H), 4.08 (t,
J = 7.3 Hz, 8H), 2.47 (p,
J = 7.3 Hz, 4H);
13C NMR (MeOD, 101 MHz) δ 156.0 (C), 144.2 (CH), 143.1 (C), 132.6 (CH), 118.1 (C), 114.1
(CH), 91.4 (CH), 52.3 (CH
2), 17.0 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 500 nm); HRMS (ESI) calcd for C
19H
20N
3 [M+H]
+ 290.1652, found 290.1650.
Example 61. 3,7-Di(azetidin-1-yl)phenoxazin-5-iumtrifluoroacetate
[0275]

[0276] Step 1: Amplex Red (449 mg, 1.75 mmol) was taken up in CH
2Cl
2 (45 mL) and cooled to 0 °C. Pyridine (1.14 mL, 14.0 mmol, 8.0 eq) and trifluoromethanesulfonic
anhydride (1.17 mL, 6.98 mmol, 4.0 eq) were added, and the ice bath was removed. The
reaction was stirred at room temperature for 3 h. It was subsequently diluted with
water and extracted with CH
2Cl
2 (2×). The combined organic extracts were washed with brine, dried (MgSO
4), filtered, and concentrated
in vacuo. Flash chromatography on silica gel (0-35% EtOAc/hexanes, linear gradient) afforded
836 mg (92%) of 10-acetyl-10H-phenoxazine-3,7-diyl bis(trifluoromethanesulfonate)
as an off-white solid.
1H NMR (CDCl
3, 400 MHz) δ 7.58 - 7.54 (m, 2H), 7.14 - 7.09 (m, 4H), 2.35 (s, 3H);
19F NMR (CDCl
3, 376 MHz) δ -73.15 (s);
13C NMR (CDCl
3, 101 MHz) δ 168.9 (C), 151.0 (C), 147.4 (C), 129.1 (C), 126.3 (CH), 118.8 (q,
1JCF = 320.9 Hz, CF
3), 117.2 (CH), 111.1 (CH), 23.0 (CH
3); HRMS (ESI) calcd for C
16H
10F
6NO
8S
2 [M+H]
+ 521.9747, found 521.9748.
[0277] Step 2: A vial was charged with the ditriflate from Step 1 (150 mg, 0.288 mmol), Pd
2dba
3 (26 mg, 0.029 mmol, 0.1 eq), XPhos (41 mg, 0.086 mmol, 0.3 eq), and Cs
2CO
3 (262 mg, 0.806 mmol, 2.8 eq). The vial was sealed and evacuated/backfilled with nitrogen
(3×). Dioxane (4 mL) was added, and the reaction was flushed again with nitrogen (3×).
Following the addition of azetidine (47 µL, 0.691 mmol, 2.4 eq), the reaction was
stirred at 80 °C for 4 h. It was then cooled to room temperature, deposited onto Celite,
and concentrated to dryness. Purification by silica gel chromatography (5-50% EtOAc/hexanes,
linear gradient; dry load with Celite) afforded 1-(3,7-di(azetidin-1-yl)-10
H-phenoxazin-10-yl)ethanone (91 mg, 94%) as a colorless solid. MS (ESI) calcd for C
20H
22N
3O
2 [M+H]
+ 336.2, found 336.2.
[0278] Step 3: The intermediate from Step 2 (63 mg, 0.189 mmol) was taken up in a mixture of CH
2Cl
2 (11.7 mL) and water (1.3 mL) and cooled to 0 °C. DDQ (47 mg, 0.207 mmol, 1.1 eq)
was added, and the reaction was stirred at room temperature for 2.5 h. A second portion
of DDQ (21 mg, 0.094 mmol, 0.5 eq) was added, and the reaction was stirred for an
additional 30 min. The mixture was evaporated, redissolved in MeCN, deposited onto
Celite, and concentrated to dryness. Silica gel chromatography (0-15% MeOH/CH
2Cl
2, linear gradient, with constant 1% v/v AcOH additive; dry load with Celite) followed
by reverse phase HPLC (10-95% MeCN/H
2O, linear gradient, with constant 0.1% v/v TFA additive) afforded 38 mg (50%) of the
title compound 3,7-di(azetidin-1-yl)phenoxazin-5-ium trifluoroacetate as a deep blue
solid.
1H NMR (MeOD, 400 MHz) δ 7.72 (d,
J = 9.3 Hz, 2H), 6.92 (dd,
J = 9.3, 2.4 Hz, 2H), 6.50 (d,
J = 2.4 Hz, 2H), 4.43 (t,
J = 7.7 Hz, 8H), 2.60 (p,
J = 7.7 Hz, 4H);
19F NMR (MeOD, 376 MHz) δ -75.45 (s);
13C NMR (MeOD, 101 MHz) δ 158.0 (C), 150.3 (C), 135.4 (CH), 135.3 (C), 116.4 (CH), 95.1
(CH), 53.7 (CH
2), 16.6 (CH
2); Analytical HPLC: >99% purity (4.6 mm × 150 mm 5 µm C18 column; 5 µL injection;
10-95% CH
3CN/H
2O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min flow;
ESI; positive ion mode; detection at 650 nm); HRMS (ESI) calcd for C
18H
18N
3O [M]
+ 292.1444, found 292.1439.
[0279] Throughout this document, various references are mentioned.
REFERENCES
[0280]
- 1 Kremers, G.-J., Gilbert, S. G., Cranfill, P. J., Davidson, M. W. & Piston, D. W. Fluorescent
proteins at a glance. J. Cell Sci. 124, 157-160, (2011).
- 2 Xia, T., Li, N. & Fang, X. Single-molecule fluorescence imaging in living cells. Ann.
Review Phys. Chem. 64, 459-480, (2013).
- 3 Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells.
Chem. Biol. 15, 128-136, (2008).
- 4 Los, G. V. et al. HaloTag: A novel protein labeling technology for cell imaging and
protein analysis. ACS Chem. Biol. 3, 373-382, (2008).
- 5 Encell, L. P. et al. Development of a dehalogenase-based protein fusion tag capable
of rapid, selective and covalent attachment to customizable ligands. Curr. Chem. Genomics
6, (Suppl 1-M7) 55-71, (2012).
- 6 Wombacher, R. et al. Live-cell super-resolution imaging with trimethoprim conjugates.
Nat. Methods 7, 717-719, (2010).
- 7 Zhao, Z. W. et al. Spatial organization of RNA polymerase II inside a mammalian cell
nucleus revealed by reflected light-sheet superresolution microscopy. Proc. Natl.
Acad. Sci. U.S.A. 111, 681-686, (2014).
- 8 Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus
microscopy. Nat. Methods 10, 60-63, (2013).
- 9 Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem
cells. Cell 156, 1274-1285, (2014).
- 10 Beija, M., Afonso, C. A. M. & Martinho, J. M. G. Synthesis and applications of rhodamine
derivatives as fluorescent probes. Chem. Soc. Rev. 38, 2410-2433, (2009).
- 11 Lavis, L.D. & Raines, R.T. Bright building blocks for chemical biology. ACS Chem.
Biol. 9, 855-866 (2014).
- 12 Grimm, J. B. et al. Carbofluoresceins and carborhodamines as scaffolds for high-contrast
fluorogenic probes. ACS Chem. Biol. 8, 1303-1310, (2013).
- 13 Arden-Jacob, J., Frantzeskos, J., Kemnitzer, N. U., Zilles, A. & Drexhage, K. H. New
fluorescent markers for the red region. Spectrochim. Acta, Part A 57, 2271-2283, (2001).
- 14 Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Evolution of Group 14 rhodamines
as platforms for near-infrared fluorescence probes utilizing photoinduced electron
transfer. ACS Chem. Biol. 6, 600-608, (2011).
- 15 Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution
microscopy of cellular proteins. Nature Chem. 5, 132-139, (2013).
- 16 Watkins, R. W., Lavis, L. D., Kung, V. M., Los, G. V. & Raines, R. T. Fluorogenic
affinity label for the facile, rapid imaging of proteins in live cells. Org. Biomol.
Chem. 7, 3969-3975, (2009).
- 17 Panchuk-Voloshina, N. et al. Alexa Dyes, a series of new fluorescent dyes that yield
exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47, 1179-1188,
(1999).
- 18 Kolmakov, K. et al. Polar red-emitting rhodamine dyes with reactive groups: Synthesis,
photophysical Properties, and two-color STED nanoscopy applications. Chem. Eur. J.
20, 146-157, (2013).
- 19 Grimm, J. B. & Lavis, L. D. Synthesis of rhodamines from fluoresceins using Pd-catalyzed
C-N cross-coupling. Org. Lett. 13, 6354-6357, (2011).
- 20 Grabowski, Z. R., Rotkiewicz, K. & Rettig, W. Structural changes accompanying intramolecular
electron transfer: Focus on twisted intramolecular charge-transfer states and structures.
Chem. Rev. 103, 3899-4032, (2003).
- 21 Vogel, M., Rettig, W., Sens, R. & Drexhage, K. H. Structural relaxation of rhodamine
dyes with different N-substitution patterns-a study of fluorescence decay times and
quantum yields. Chem. Phys. Lett. 147, 452-460, (1988).
- 22 Song, X., Johnson, A. & Foley, J. 7-Azabicyclo[2.2.1]heptane as a unique and effective
dialkylamino auxochrome moiety: Demonstration in a fluorescent rhodamine dye. J. Am.
Chem. Soc 130, 17652-17653, (2008).
- 23 Rozeboom, M. D., Houk, K., Searles, S. & Seyedrezai, S. E. Photoelectron spectroscopy
of N-aryl cyclic amines. Variable conformations and relationships to gas-and solution-phase
basicities. J. Am. Chem. Soc. 104, 3448-3453, (1982).
- 24 Karstens, T. & Kobs, K. Rhodamine B and rhodamine 101 as reference substances for
fluorescence quantum yield measurements. J. Phys. Chem. 84, 1871-1872, (1980).
- 25 Cavallo, L., Moore, M. H., Corrie, J. E. T. & Fratemali, F. Quantum mechanics calculations
on rhodamine dyes require inclusion of solvent water for accurate representation of
the structure. J. Phys. Chem. A 108, 7744-7751, (2004).
- 26 Pearson, W. H., Lian, B. W. & Bergmeier, S. C. in Comprehensive Heterocyclic Chemistry
II Vol. 1A (eds A. R. Katritzky, C. W. Rees, & E. F. V. Scriven) 1 (Elsevier, 1996).
- 27 Smith, S. A., Hand, K. E., Love, M. L., Hill, G. & Magers, D. H. Conventional strain
energies of azetidine and phosphetane: Can density functional theory yield reliable
results? J. Comp. Chem. 34, 558-565, (2013).
- 28 Mütze, J. et al. Excitation spectra and brightness optimization of two-photon excited
probes. Biophys. J. 102, 934-944, (2012).
- 29 Neklesa, T. K. et al. Small-molecule hydrophobic tagging-induced degradation of HaloTag
fusion proteins. Nat. Chem. Biol. 7, 538-543, (2011).
- 30 Zhang, Z., Revyakin, A., Grimm, J. B., Lavis, L. D. & Tjian, R. Single-molecule tracking
of the transcription cycle by sub-second RNA detection. eLife 3, e01775, (2014).
- 31 Wu, B., Chao, J. A. & Singer, R. H. Fluorescence fluctuation spectroscopy enables
quantitative imaging of single mRNAs in living cells. Biophys. J. 102, 2936-2944,
(2012).
- 32 Mchedlov-Petrossyan, N., Vodolazkaya, N. & Doroshenko, A. Ionic equilibria of fluorophores
in organized solutions: The influence of micellar microenvironment on protolytic and
photophysical properties of rhodamine B. J. Fluoresc. 13, 235-248, (2003).
- 33 Bancaud, A. et al. Molecular crowding affects diffusion and binding of nuclear proteins
in heterochromatin and reveals the fractal organization of chromatin. EMBO J. 28,
3785-3798, (2009).
- 34 Bancaud, A., Lavelle, C., Huet, S. & Ellenberg, J. A fractal model for nuclear organization:
Current evidence and biological implications. Nucleic Acids Res. 40, 8783-8792, (2012).
- 35 Altman, R. B. et al. Cyanine fluorophore derivatives with enhanced photostability.
Nat. Methods 9, 68-71, (2012).
- 36 Egawa, T.; Koide, Y.; Hanaoka, K.; Komatsu, T.; Terai, T.; Nagano, T. Chem. Commun.
2011, 47, 4162-4164.
- 37 Suzuki, K.; Kobayashi, A.; Kaneko, S.; Takehira, K.; Yoshihara, T.; Ishida, H.; Shiina,
Y.; Oishi, S.; Tobita, S. Phys. Chem. Chem. Phys. 2009, 11, 9850-9860.
- 38 Akerboom, J.; Chen, T.-W.; Wardill, T. J.; Tian, L.; Marvin, J. S.; Mutlu, S.; Calderón,
N. C.; Esposti, F.; Borghuis, B. G.; Sun, X. R. J. Neurosci. 2012, 32, 13819-13840.
- 39 Magde, D., Rojas, G.E. & Seybold, P.G. Solvent dependence of the fluorescence lifetimes
of xanthene dyes. Photochem. Photobiol. 70, 737-744 (1999).
- 40 Lavis, L.D., Rutkoski, T.J. & Raines, R.T. Tuning the pKa of fluorescein to optimize
binding assays. Anal. Chem. 79, 6775-6782 (2007).
- 41 Revyakin, A.; Zhang, Z.; Coleman, R. A.; Li, Y.; Inouye, C.; Lucas, J. K.; Park, S.-R.;
Chu, S.; Tjian, R. Genes Dev. 2012, 26, 1691-1702.
- 42 Arnauld, S.; Nicolas, B.; Hervé, R.; Didier, M. Nature Protocol Exchange 2008, doi:10.1038/nprot.2008.128.
- 43 Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant
protein molecules inside live cells. Science 281, 269-272 (1998).
- 44 Keppler, A. et al. A general method for the covalent labeling of fusion proteins with
small molecules in vivo. Nat. Biotechnol. 21, 86-89 (2002).
- 45 Hori, Y., Ueno, H., Mizukami, S. & Kikuchi, K. Photoactive yellow protein-based protein
labeling system with turn-on fluorescence intensity. J. Am. Chem. Soc. 131, 16610-16611
(2009).
- 46 Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside
living cells. Proc. Natl. Acad. Sci. U.S.A. 107, 10914-10919 (2010).
- 47 Testa, I. et al. Multicolor fluorescence nanoscopy in fixed and living cells by exciting
conventional fluorophores with a single wavelength. Biophys. J. 99, 2686-2694 (2010).
- 48 Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J. & Waggoner, A.S. Cyanine
dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjugate Chem. 4,
105-111 (1993).
- 49 Haugland, R.P., Spence, M.T.Z., Johnson, I.D. & Basey, A. The Handbook: A Guide to
Fluorescent Probes and Labeling Technologies, 10th ed., (Molecular Probes, 2005).
- 50 Bosch, P.J. et al. Evaluation of fluorophores to label SNAP-tag fused proteins for
multicolor single-molecule tracking microscopy in live cells. Biophys. J. 107, 803-814
(2014).
- 51 Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional
fluorescent probes. Angew. Chem. Int. Ed. 47, 6172-6176 (2008).
- 52 Dempsey, G.T., Vaughan, J.C., Chen, K.H., Bates, M. & Zhuang, X. Evaluation of fluorophores
for optimal performance in localization-based super-resolution imaging. Nat. Methods
8, 1027-1036 (2011).
- 53 Ha, T. & Tinnefeld, P. Photophysics of fluorescence probes for single molecule biophysics
and super-resolution imaging. Annu. Rev. Phys. Chem. 63, 595-617 (2012).
- 54 Lukinavič̌̌ius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton.
Nat. Methods 11, 731-733 (2014).
- 55 Kubota, Y. & Steiner, R.F. Fluorescence decay and quantum yield characteristics of
acridine orange and proflavine bound to DNA. Biophys. Chem. 6, 279-289 (1977).
- 56 Lee, L.G., Berry, G.M. & Chen, C.-H. Vita Blue: A new 633-nm excitable fluorescent
dye for cell analysis. Cytometry 10, 151-164 (1989).
- 57 Speight, L.C. et al. Efficient synthesis and in vivo incorporation of acridon-2-ylalanine,
a fluorescent amino acid for lifetime and Forster resonance energy transfer/luminescence
resonance energy transfer studies. J. Am. Chem. Soc. 135, 18806-18814 (2013).
- 58 Mitronova, G.Y. et al. New fluorinated rhodamines for optical microscopy and nanoscopy.
Chem. Eur. J. 16, 4477-4488 (2010).
- 59 Critchfield, F.E., Gibson Jr, J.A. & Hall, J.L. Dielectric constant for the dioxane-water
system from 20 to 35°. J. Am. Chem. Soc. 75, 1991-1992 (1953).
- 60 Mütze, J. et al. Excitation spectra and brightness optimization of two-photon excited
probes. Biophys. J. 102, 934-944 (2012).
- 61 Lavis, L.D. & Raines, R.T. Bright ideas for chemical biology. ACS Chem. Biol. 3, 142-155
(2008).
- 62 Kövér, J. & Antus, S. Facile deoxygenation of hydroxylated flavonoids by palladium-catalysed
reduction of its triflate derivatives. Z. Naturforsch., B: J. Chem. Sci. 60, 792-796
(2005).
- 63 Urgaonkar, S. & Verkade, J.G. Palladium/proazaphosphatrane-catalyzed amination of
aryl halides possessing a phenol, alcohol, acetanilide, amide or an enolizable ketone
functional group: efficacy of lithium bis(trimethylsilyl)amide as the base. Adv. Synth.
Catal. 346, 611-616 (2004).
- 64 Whitaker, J.E. et al. Fluorescent rhodol derivatives: Versatile, photostable labels
and tracers. Anal. Biochem. 207, 267-279 (1992).
- 65 Sauers, R.R., Husain, S.N., Piechowski, A.P. & Bird, G.R. Shaping the absorption and
fluorescence bands of a class of efficient, photoactive chromophores: synthesis and
properties of some new 3H-xanthen-3-ones. Dyes Pigments 8, 35-53 (1987).